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Correlation of the level of β-citryl-L-glutamic acid with spermatogenesis in rat testis
Abstract
beta-Citryl-L-glutamic acid, which is known to be highly concentrated in the brains of immature animals, is preferentially localized in the testes of various adult animals, including mammals, amphibians and fish, mainly in the germinal cells. In young rats, the citrylglutamate concentration increases with age and coincides with the development of late spermatocytes into early spermatids. Rats with seminiferous tubule failure induced by ductuli efferentes ligation and experimental cryptorchidism are infertile as a result of germ cell depletion, especially spermatocytes and early spermatids. In these animals, the testicular citrylglutamate content was much lower than in normal testes.
... In the rat nervous system, bCG level is highest at birth and then continuously decreases during postnatal development [15]. In the adult, bCG is present in brain and various other organs at concentrations in the range of 5 to 50 nmol/g wet weight [16]. The highest bCG concentrations are, however, found in the adult testis (high mM to mM range) of ( possibly all) vertebrates [16], suggesting an important role of this pseudo peptide in spermatogenesis. ...
... In the adult, bCG is present in brain and various other organs at concentrations in the range of 5 to 50 nmol/g wet weight [16]. The highest bCG concentrations are, however, found in the adult testis (high mM to mM range) of ( possibly all) vertebrates [16], suggesting an important role of this pseudo peptide in spermatogenesis. bCG is synthesized by an amino acid ligase, bCG synthase (CGS), an enzyme encoded by the gene Rimklb [17]. ...
... The developmental increase in bCG concentration in testis exhibits a close correlation in time with the first appearance of spermatids [16]. Analysis of bCG content suggested that high bCG concentrations are present in germ cells ( particularly in late spermatocytes and early spermatids) and Rimklb gene expression is up-regulated in the meiotic prophase I in leptotene/zygotene spermatocytes [24]. ...
Chromatin remodelling in spermatids is an essential step in spermiogenesis and involves the exchange of most histones by protamines, which drives chromatin condensation in late spermatids. The gene Rimklb encodes a citrylglutamate synthase highly expressed in testes of vertebrates and the increase of its reaction product, β-citrylglutamate, correlates in time with the appearance of spermatids. Here we show that deficiency in a functional Rimklb gene leads to male subfertility, which could be partially rescued by in vitro fertilization. Rimklb-deficient mice are impaired in a late step of spermiogenesis and produce spermatozoa with abnormally shaped heads and nuclei. Sperm chromatin in Rimklb-deficient mice was less condensed and showed impaired histone to protamine exchange and retained transition protein 2. These observations suggest that citrylglutamate synthase, probably via its reaction product β-citrylglutamate, is essential for efficient chromatin remodelling during spermiogenesis and may be a possible candidate gene for male subfertility or infertility in humans.
... N α -Acetyl conjugates for all 20 of the common amino acids have been identified in mammals. In addition, the N α -acetyl conjugates of other amino acids, including β-alanine, allo-isoleucine, α-aminobutyric acid, GABA, 2-aminooctanoic acid, citrulline and N ε -acetyllysine have also been characterized from mammalian sources [48][49][50][51][52][53][54][55][56][57][58][59][60][61]. With the exception of N-acetylglutamate, which serves as an allosteric activator of carbamoyl phosphate synthetase I [62], the N-acetylamino acid conjugates are trace metabolites that function in the excretion/detoxification of abnormally high levels of a particular amino acid. ...
... [69][70]Bile acid conjugation to glycine or taurine increases bile acid solubility, renders the bile acids impermeable to cell membranes and is essential to proper liver function [69]. In addition, β-citrylglutamate may have a role in spermatogenesis [54] and in the differentiation of lens epithelial cells into fiber cells [70]. ...
... Alanine Arachidonoyl [76] γ-Aminobutyric acid Arachidonoyl [76] Glutamic Acid β-Citryl and phenylacetyl [54,55,114] Glutamine Phenylacetyl, other arylacetyls, and 4phenylbutyryl [55,114] Glycine c Arachidonoyl, benzoyl, butyryl, bile acids, decanoyl, hexanoyl, isobutyryl, 2-methylbutyryl, 3-methylcrotonyl, octanoyl, phenylacetyl and other arylacetyls, propionyl, suberyl, and tiglyl [7,55,57,65,76,84,115] Isoleucine Lactyl [116] Leucine Lactyl [116] Phenylalanine Succinoyl [117] Pyroglutamic acid Phenylacetyl [57] Serine Arachidonoyl [71] Taurine Bile acids, phenylacetyl and other arylacetyls, long chain, saturaturated acyl groups from C16:0-C26:0 d , long-chain, monounsaturated acyl groups from C18:1-C24:1 d [7,84,118] Valine Lactyl [116] a N-Acetyl and N-isovaleroylamino acids were not included in this table. ...
The identification of two biologically active fatty acid amides, N-arachidonoylethanolamine (anandamide) and oleamide, has generated a great deal of excitement and stimulated considerable research. However, anandamide and oleamide are merely the best-known and best-understood members of a much larger family of biologically occurring fatty acid amides. In this review, we will outline which fatty acid amides have been isolated from mammalian sources, detail what is known about how these molecules are made and degraded in vivo, and highlight their potential for the development of novel therapeutics.
... In primary cultures of neurons from newborn mouse brain, BCG, which was suggested to serve as an Fe-carrier for aconitase [44], enhanced cell viability by accelerating mitochondrial activity. Rats that are infertile due to germ cell depletion show low beta-citrylglutamate concentrations, suggesting its involvement in the metabolic support of cell proliferation [46]. BCG is a physiological substrate of the enzyme beta-citrylglutamate synthase-B, encoded by the RIMKLB gene [47]. ...
Resistance training promotes metabolic health and stimulates muscle hypertrophy, but the precise routes by which resistance exercise (RE) conveys these health benefits are largely unknown.
Aim:
To investigate how acute RE affects human skeletal muscle metabolism.
Methods:
We collected vastus lateralis biopsies from six healthy male untrained volunteers at rest, before the first of 13 RE training sessions, and 45 min after the first and last bouts of RE. Biopsies were analysed using untargeted mass spectrometry-based metabolomics.
Results:
We measured 617 metabolites covering a broad range of metabolic pathways. In the untrained state RE altered 33 metabolites, including increased 3-methylhistidine and N-lactoylvaline, suggesting increased protein breakdown, as well as metabolites linked to ATP (xanthosine) and NAD (N1-methyl-2-pyridone-5-carboxamide) metabolism; the bile acid chenodeoxycholate also increased in response to RE in muscle opposing previous findings in blood. Resistance training led to muscle hypertrophy, with slow type I and fast/intermediate type II muscle fibre diameter increasing by 10.7% and 10.4%, respectively. Comparison of post-exercise metabolite levels between trained and untrained state revealed alterations of 46 metabolites, including decreased N-acetylated ketogenic amino acids and increased beta-citrylglutamate which might support growth. Only five of the metabolites that changed after acute exercise in the untrained state were altered after chronic training, indicating that training induces multiple metabolic changes not directly related to the acute exercise response.
Conclusion:
The human skeletal muscle metabolome is sensitive towards acute RE in the trained and untrained states and reflects a broad range of adaptive processes in response to repeated stimulation.
... Potentially, BCG might be important for brain development, as it was found in high concentrations in the newborn rat brain but disappeared with maturation (152,153). Similarly, BCG was detected in the germinal cells of the rat testes, and its amount increased with the appearance of late spermatocytes and early spermatids (154), suggesting it could be involved in spermatogenesis. Follow-up studies linked BCG to physiologically important chelation of iron and copper (155,156). ...
Glutamate carboxypeptidases II and III (GCPII and GCPIII) are highly homologous di-zinc metallopeptidases belonging to the M28 family. These enzymes are expressed in a variety of tissues, including the brain, prostate, kidney, testis and jejunum. GCPII has been recognized as a neuropeptidase in the central nervous system, as a folate hydrolase participating in absorption of folates in the jejunum and, most importantly, as a prostate-specific membrane antigen that is highly expressed in prostate adenocarcinoma. Furthermore, it has been identified in the neovasculature of most human solid tumors. In contrast, GCPIII has not been associated with any specific physiological function or pathology, and its expression, activity and inhibition have not been as well-studied. In this review, we provide an overview of the current understanding of the structure, enzymatic activity, substrate specificity, and tissue distribution of these two homologous enzymes. We discuss their potential physiological functions and describe the available animal models, including genetically modified mice. We also review the potential use of specific monoclonal antibodies and small-molecule inhibitors recognizing GCPII/III for diagnosis, imaging and experimental therapy of human cancers and other pathologies.
... Despite these extremely high brain concentrations, the function of BCG is elusive. Thus far, BCG has been suggested to be related to cell differentiation (62)(63)(64)(65) and neuronal protection by metal chelation (66 -68). Because we are the first to report the existence of BCG 2 , its function, if any, is unclear. ...
The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain
barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5−/− mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter
N-acetylaspartylglutamate was 2.4-fold higher in Abcc5−/− brain. The metabolites that accumulated in Abcc5−/− tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also
transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the
therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs
are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter
in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5−/− brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate
conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs.
... Similarly, high levels of glutathione and the synthetic machinery to produce it are present in the testis, 59 particularly in cells such as spermatogonia, spermatocytes and Sertoli cells. Other glutamate-like compounds such as citryl-L-glutamic acid are known to be highly enriched in the testis, 60 but it is unclear where they are localized and how they relate metabolically to glutamate. Figure 12 Immunocytochemistries for endogenous glutamate and for D-aspartate. ...
Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, western blotting and immunocytochemistry the expression of a panel of sodium-dependent plasmalemmal glutamate transporters in the rat testis. Proteins examined included: glutamate aspartate transporter (GLAST), glutamate transporter 1 (GLT1), excitatory amino acid carrier 1 (EAAC1), excitatory amino acid transporter 4 (EAAT4) and EAAT5. We demonstrate that many of the glutamate transporters in the testis are alternately spliced. GLAST is present as exon-3- and exon-9-skipping forms. GLT1 was similarly present as the alternately spliced forms GLT1b and GLT1c, whereas the abundant brain form (GLT1a) was detectable only at the mRNA level. EAAT5 was also strongly expressed, whereas EAAC1 and EAAT4 were absent. These patterns of expression were compared with the patterns of endogenous glutamate localization and with patterns of d-aspartate accumulation, as assessed by immunocytochemistry. The presence of multiple glutamate transporters in the testis, including unusually spliced forms, suggests that glutamate homeostasis may be critical in this organ. The apparent presence of many of these transporters in the testis and sperm may indicate a need for glutamate transport by such cells.
Resistance training promotes metabolic health and stimulates muscle hypertrophy, but the precise routes by which resistance exercise (RE) conveys these health benefits is largely unknown. Aim: To investigate how acute RE affects human skeletal muscle metabolism. Methods: We collected vastus lateralis biopsies from six healthy male untrained volunteers at rest, before the first of 13 RE training sessions, and 45 min after the first and last bouts of RE. Biopsies were analysed using untargeted mass spectrometry-based metabolomics. Results: We measured 617 metabolites covering a broad range of metabolic pathways. In the untrained state RE altered 33 metabolites, including increased 3-methylhistidine and 1-carboxylethylvaline, suggesting increased protein breakdown, as well as metabolites linked to ATP (xanthosine) and NAD (N1-methyl-2-pyridone-5-carboxamide) metabolism; the bile acid chenodeoxycholate also increased in response to RE in muscle opposing previous findings in blood. Resistance training led to muscle hypertrophy, with slow type I and fast/intermediate type II muscle fibre diameter increasing by 10.7% and 10.4%, respectively. Comparison of post-exercise metabolite levels between trained and untrained state revealed alterations of 46 metabolites, including decreased N-acetylated ketogenic amino acids and increased beta-citrylglutamate which might support growth. Only five of the metabolites that changed after acute exercise in the untrained state were altered after chronic training, indicating that training induces multiple metabolic changes not directly related to the acute exercise response. Conclusion: The human skeletal muscle metabolome is sensitive towards acute RE in the trained and untrained states and reflects a broad range of adaptive processes in response to repeated stimulation.
Glutamate carboxypeptidases II and III (GCPII and GCPIII) are highly homologous di-zinc metallopeptidases belonging to the M28 family. These enzymes are expressed in a variety of tissues, including the brain, prostate, kidney, testis and jejunum. GCPII has been recognized as a neuropeptidase in the central nervous system, as a folate hydrolase participating in absorption of folates in the jejunum and, most importantly, as a prostate-specific membrane antigen that is highly expressed in prostate adenocarcinoma. Furthermore, it has been identified in the neovasculature of most human solid tumors. In contrast, GCPIII has not been associated with any specific physiological function or pathology, and its expression, activity and inhibition have not been as well-studied. In this review, we provide an overview of the current understanding of the structure, enzymatic activity, substrate specificity, and tissue distribution of these two homologous enzymes. We discuss their potential physiological functions and describe the available animal models, including genetically modified mice. We also review the potential use of specific monoclonal antibodies and small-molecule inhibitors recognizing GCPII/III for diagnosis, imaging and experimental therapy of human cancers and other pathologies.
Unlabelled:
Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave β-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body.
Database:
The X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09.
We present here a structure-aided design of inhibitors targeting the active site as well as exosites of glutamate carboxypeptidase II (GCPII), a prostate cancer marker, preparing potent and selective inhibitors that are more than 1000-fold more active toward GCPII than its closest human homolog, glutamate carboxypeptidase III (GCPIII). Additionally, we demonstrate that the prepared inhibitor conjugate can be used for sensitive and selective imaging of GCPII in mammalian cells.
Developmental changes in the concentration of β‐citryl‐L‐glutamate (β‐CG) have been examined in the cerebrum and optic lobe of the developing chick brain and in primary cultured neuronal cells from the chick embryo optic lobes with gas chromatographic and HPLC methods originated in our studies. A sharp peak was shown by β‐CG, with a maximal concentration at 13 days of incubation in the optic lobe of the developing chick brain but decreasing markedly to adult levels. The developmental change in primary cultured neurons was similar to that in the optic lobe of the developing chick brain. Changes in synthetic and hydrolytic activities of β‐CG were studied during growth of primary cultured neurons. Incorporation of radioactivities from radiolabeled pyruvate and alanine into β‐CG increased significantly on day 3 of culture, reaching a plateau on day 6, whereas that from radioactive glutamine and glutamate increased gradually from day 3 to day 12 of culture. The hydrolyzing enzyme activity of β‐CG during neuron growth was low until day 3 of culture, when it increased significantly until day 12. Similar developmental changes were observed in the developing chick embryo optic lobes.
ß-Citryl-l-glutamate-hydrolysing enzyme (ß-CGHE) was purified from rat testis particulate fraction 13 000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, ß-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed ß-citryl-l-glutamate (ß-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the ß-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. ß-CGHE was also potently inhibited by an excitatory amino acid agonist, l-quisqualate, but not by another agonists, and kinate. It had high substrate specificity for ß-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.
β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.
The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and β-citrylglutamate,
a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized
on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched
the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode
two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively
expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both
proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and l-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of β-citrylglutamate. The nature of the reaction products
was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement
with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress
in the understanding of the function of NAAG and β-citrylglutamate.
The compound beta-citryl-L-glutamate (beta-CG) was initially isolated from developing brains, while it has also been found in high concentrations in testes and eyes. However, its functional roles are unclear. To evaluate its coordination with metal ions, we performed pH titration experiments. The stability constant, logbeta(pqr) for M(p)(beta-CG)(q)H(r) was calculated from pH titration data, which showed that beta-CG forms relatively strong complexes with Fe(III), Cu(II), Fe(II) and Zn(II). beta-CG was also found able to solubilize Fe more effectively from Fe(OH)(2) than from Fe(OH)(3). Therefore, we examined the effects of beta-CG on Fe-dependent reactive oxygen species (ROS)-generating systems, as well as the potential ROS-scavenging activities of beta-CG and metal ion-(beta-CG) complexes. beta-CG inhibited the Fe-dependent degradation of deoxyribose and Fe-dependent damage to DNA or plasmid DNA in a dose-dependent manner, whereas it had no effect on Cu-mediated DNA damage. In addition, thermodynamic data showed that beta-CG in a physiological pH solution is an Fe(II) chelator rather than an Fe(III) chelator. Taken together, these findings suggest that beta-CG is an endogenous low molecular weight Fe chelator.
Developmental changes in the concentration of beta-citryl-L-glutamate(beta-CG) have been examined in the cerebrum and optic lobe of the developing chick brain and in primary cultured neuronal cells from the chick embryo optic lobes with gas chromatographic and HPLC methods originated in our studies. A sharp peak was shown by beta-CG, with a maximal concentration at 13 days of incubation in the optic lobe of the developing chick brain but decreasing markedly to adult levels. The developmental change in primary cultured neurons was similar to that in the optic lobe of the developing chick brain. Changes in synthetic and hydrolytic activities of beta-CG were studied during growth of primary cultured neurons. Incorporation of radioactivities from radiolabeled pyruvate and alanine into beta-CG increased significantly on day 3 of culture, reaching a plateau on day 6, whereas that from radioactive glutamine and glutamate increased gradually from day 3 to day 12 of culture. The hydrolyzing enzyme activity of beta-CG during neuron growth was low until day 3 of culture, when it increased significantly until day 12. Similar developmental changes were observed in the developing chick embryo optic lobes.
An enzyme responsible for the deacylation of beta-citryl-L-glutamate to citrate and glutamate has been characterized in rat testis. The enzyme required manganese ion for full activity and was strongly inhibited by nucleotides such as ATP or GTP. The activity was localized in the particulate fractions. The enzyme favored N-formyl-L-glutamate greater than beta-citrly-L-glutamate greater than beta-citryl-L-glutamine in a decreasing order. The amidohydrolyase activity was highest in the testis and lung, a moderate activity was detected in heart, kidney and intestine, and low in brain, thymus, stomach, skeletal muscle, spleen and liver. These findings suggest that the amidohydrolase is different from any of amidohydrolases reported so far, amidohydrolase I (EC 3.5.1.14), II (EC 3.5.1.15), III, N-acetyl-lysine deacylase (EC 3.5.1.17) and N-acetyl-beta-alanine deacetylase (EC 3.5.1.21), and various peptidases.
The beta-CG concentration in the chicken brain was high during embryonic development and decreased rapidly to a lower level close to hatching, while the concentration in the eyeball which was also high during the embryonic life retained a fairly high level after hatching. The distribution of beta-CG in the bovine eye was determined. About 95% of total beta-CG content in the whole eye was localized in the lens. However, the distribution of beta-CG in the eye varied depending on species. beta-CG was exclusively localized in the lens in the eyes of fish and mammals, but distributed in both lens and retina in frogs. The molecule was localized in the retina rather than the lens in the chicken eye, although the concentrations was extremely low compared to those in the mammalian, amphibian and fish eyes. It was found that beta-CG is present ubiquitously in the lens or retina in various species. The distribution of beta-CG in the bovine lens was determined in the three cortex regions and nucleus. beta-CG was present at the highest concentration in the equatorial cortex, at a moderate concentration in the posterior and anterior cortex, and at the lowest concentration in the nucleus. Similar distribution patterns were also found in the rabbit and rat lens. When embryonic chick lens epithelial cells were cultured in the presence of fetal calf serum, the cells elongated, differentiated into fiber cells and formed lentoid bodies. The cells of lentoid bodies were stained strongly by the anti-beta-CG antibody, while cells around the structures were not. In addition, the beta-CG content in the lenses from the galactose cataractous rat decreased to about 20-30% of that in the normal lens. These findings suggest that beta-CG may play a role in the differentiation of epithelial cells into fiber cells.
Beta-citryl-L-glutamate (beta-CG) concentration was determined by HPLC during the differentiation of bovine lens epithelial cells into lens fiber cells in culture. beta-CG increased from 1 to 4 weeks of culture and then decreased slightly, while alpha-crystallin, a marker of lens cell differentiation, increased rapidly 4 weeks after the culture and continued to increase gradually until week 11. In addition, the localization of beta-CG was immunohistochemically examined using anti-beta-CG antibody. Cells around lentoid bodies were stained with anti-beta-CG antibody, whereas cells in the bodies were stained strongly with anti-gamma-crystallin antibody. These findings suggest that beta-CG accumulated immediately before the differentiation of the bovine lens epithelial cells into lens fiber cells and may play a role in regulating the differentiation of lens cells.
The immunocytochemical localization of beta-citryl-L-glutamate (beta-CG) in primary neuronal cells and in the differentiation of P19 cells was examined. 1: Cells with the morphological features of neurons in the primary culture were specifically stained with the anti-beta-CG antibody both in neurites and in the cell body. 2: The neuronal cells differentiated from P19 cells were distinctly stained with the anti-beta-CG antibody both in neurites and in the cell body, while the non-neuronal cells were not. 3: The concentration of beta-CG was low in the P19 cells, but increased significantly with the differentiation of P19 cells into neurons. It was shown that beta-CG was localized exclusively in neurons. These findings suggest that beta-CG plays functional roles in the differentiation and growth of neuron.
A cDNA encoding rat homologue of the previously characterized mouse Sox6 was isolated by a polymerase chain reaction (PCR) cloning strategy. Comparison of this eDNA with homologous mouse, human and rainbow trout cDNA exhibited an overall amino acid sequence identity of 99.6, 89.3 and 76.3% respectively. The leucine-zipper and HMG-box motif were almost completely conserved between these homologues. The expression of Sox6 was determined in rat by Northern hybridization and Real-time quantitative reverse transcription (RT)-PCR. rSox6 (rat Sox6) was specifically expressed in the neonatal brain and adult testis with Northern blotting. Real-time quantitative RT-PCR for the determination of Sox6 mRNA was examined. The rSox6 was expressed in the neonatal brain and adult testis as well as by Northern blotting and also expressed in the adult eyeball and slightly in the ovary.
Abstract The developmental changes of N-acetylaspartic acid (NA-Asp), N-acetyl--aspartylglutamic acid (NA-Asp-Glu), and β-citryl-L-glutamic acid (β-CG) have been examined in the cerebrum, cerebellum, brain stem and spinal cord of both rat and guinea pig by the gas chromatographic method developed in our studies. A rapid increase in the concentration of NA-Asp was observed postnatally in every region of the rat brain. On the other hand, all regions of guinea pig brain showed the prenatal increases. NA-Asp-Glu showed a different developmental profile, depending on region of the brain, in the two species. The concentration of NA-Asp-Glu remained constantly low during brain maturation in the rostral regions. In the caudal portions it showed a marked increase during maturation and reached a high level in the adult brain. The concentration of β-CG was highest at birth in all regions of rat brain and rapidly decreased by 20 days after birth and remained low thereafter. The rapid decrease occurred in the guinea pig during the foetal period, and β-CG content decreased to an adult level at birth.
A simple and sensitive gas-chromatographic method for the determination of N-acetyl-l-aspartic acid (NA-Asp), N-acetyl-α-aspartylglutamic acid (NA-Asp-Glu) and β-citryl-l-glutamic acid (β-CG) was developed. The organ, regional and phylogenetic distributions of these compounds were studied. NA-Asp and NA-Asp-Glu were highly concentrated in nervous tissue, and less than 1% of the amounts in the nervous tissues were found in nonnervous organs. These two compounds showed a reciprocal relationship in their regional distribution in mature brains, but such a relationship was not evident or was even reversed in immature brains. The two compounds also showed different developmental changes in different regions of the brain. Fish brain contained a relatively high concentration of NA-Asp, but only a trace amount of NA-Asp-Glu. By contrast, a 10 times higher concentration of NA-Asp-Glu than NA-Asp was found in frog brain. Reptilian brain contained similar amounts of each compound. Avian and mammalian brain had NA-Asp at a roughly 10 times higher concentration than NA-Asp-Glu. β-CG occurred at the highest concentration in the immature brain of rat and guinea pig, but disappeared in the mature brains. The adult frog brain, however, contained a large amount of β-CG. In the adult rat, testis contained the highest concentration of β-CG.
Ab unknwon compound containing glutamic acid residue was found in newborn rat brain. The compound occurred predominantly in brain. Its concentration was approx. 1 μmol/g tissue at birth and decreased to one-tenth 24 days after birth.The compound was isolated from newborn rat brains, and subjected to elementary analysis and to infrared and mass spectrometric analysis. Glutamic acid and citric acid were formed from the compound on acid hydrolysis. The compound was presumed to be a citryglutamic acid.Two isomers, α- and β-citrylglutamic acid, were sunthesized. The unknown compound was identified as β-citryl-L-glutamic acid. The occurrence of this compound has not been reported in nature.
The activities of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase have been correlated with spermidine content and morphological criteria of cell differentiation during hormone-dependent spermatogenesis in the rat. The specific activity of ornithine decarboxylase was maximal at 3 days of age at a time when only Sertoli cells were present in the seminiferous tubules, but the specific activity was markedly reduced with the formation of additional cell types. The specific activity of S-adenosyl-L-methionine decarboxylase increased steadily during spermatogenesis, reaching a plateau with spermatozoan maturation. Spermidine concentration generally paralleled the specific activity of ornithine decarboxylase, and evidenced an increase during spermatid maturation. Hypophysectomy prevented these characteristic developmental enzyme changes and reduced testis spermidine content. Stimulation with daily injections of FSH and LH or of testosterone maintained the normal developmental pattern and the sp...
Endogenous concentrations of testosterone (T), 5α (androstan)3α, 17β diol (DIOL) and dihydrotestosterone (DHT) were determined in whole testis, seminiferous tubules and interstitial tissue of rats from 10 to 90 days of age. Contents in whole testis were very close to the sum of the contents in seminiferous tubules and interstitial tissue. In seminiferous tubules the concentration of T increased from 39 ng/100 mg proteins at 14 days to 94 at 20 days, and then dropped to 13 at 26 days. There was a gradual increase thereafter to reach again a value of 94 at sexual maturation. The concentration of DIOL also increased from 21 to 89 in 20 day old rats, but it remained high in 26 day old animals, only to drop thereafter and remain low through sexual maturation. The concentration of DHT did not show a definite peak in young rats but increased markedly between 60 and 90 days of life. The concentrations of T in interstitial tissue were much higher than in seminiferous tubules at all ages; values were between 800 and 900 in young rats and increased to approximately 4000 ng/100 mg proteins in adult animals. DIOL and DHT could not be detected in this tissue. It is speculated that T and DIOL might be the active androgens in the seminiferous tubules at the time of meiosis while T and DHT might be preponderant in rats with mature spermatogenesis.
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The effects of ligation of the vasa efferentia and vasectomy were studied in adult rats, using the periodic acid-fuchsin sulphurous acid staining technique for the identification of stages of the 'seminiferous cycle'.
The operations were performed unilaterally, the testis and the efferent ducts on the contralateral side serving as controls.
The animals were killed at intervals ranging from 3 hr. to 28 days after ligation of the vasa efferentia, and 3 hr. and 90 days after vasectomy.
Ligation of the vasa efferentia was followed by an increase in the diameter of the seminiferous tubules and in testicular weight which reached its peak at 36 hr. Thereafter both decreased progressively, the shrinkage being associated with degeneration of the seminiferous epithelium. Thus by 28 days after operation the weight of the treated testis was halved and the seminiferous epithelium was limited to a single row of cells.
The pattern of degeneration of germ cells largely depended on their developmental stage: spermatocytes and spermatids showed signs of more severe damage than spermatogonia or mature spermatozoa. The changes among the spermatocytes and spermatids similarly varied according to their developmental stage.
Vasectomy did not affect the process of spermatogenesis. The operation was followed by a temporary increase in the weight of the epididymis and by the formation of spermatoceles on the cut testicular end of the vas deferens.