Regulation of the Ecto-Nucleotidase Pathway in Rat Hippocampal Nerve Terminals

Ecto-nucleotidases play a pivotal role in terminating the signalling via ATP and in producing adenosine, a neuromodulator in the nervous system. We have now investigated the pattern of adenosine formation with different concentrations of extracellular ATP in rat hippocampal nerve terminals. It was found that adenosine formation is delayed with increasing concentrations of ATP. Also, the rate of adenosine formation increased sharply when the extracellular concentrations of ATP + ADP decrease below 5 M, indicating that ATP/ADP feed-forwardly inhibit ecto-5-nucleotidase allowing a burst-like formation of adenosine possibly designed to activate facilitatory A_2A receptors. Initial rate measurements of ecto-5-nucleotidase in hippocampal nerve terminals, using IMP as substrate, showed that ATP and ADP are competitive inhibitors (apparent K_i of 14 and 4 M). In contrast, in hippocampal immunopurified cholinergic nerve terminals, a burst-like formation of adenosine is not apparent, suggesting that channelling processes may overcome the feed-forward inhibition of ecto-5-nucleotidase, thus favouring A₁ receptor activation.     

Characterization of Sertoli cell RNA synthetic activities in vitro at selected times during sexual maturation

In this study, Sertoli cell RNA synthetic activity in vitro was characterized at selected times during sexual maturation. Sertoli cells, isolated from rat testes undergoing the first wave of spermatogenesis and placed in culture for 4 days, exhibited 2-fold increases in soluble ribonucleotide pools and in total RNA concentrations over the age span of 18-35 days. High performance liquid chromatographic analysis of the ribonucleotide pools in Sertoli cells cultured from 18- and 33- to 34-day-old rats revealed that, in addition to the overall age-related doubling of concentrations, uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools were disproportionately increased 4- and 6-fold, respectively. In general, Sertoli cell contained relatively small amounts of UTP in comparison to several other cell types, but exhibited a high ADP:ATP ratio. A uniform 2-fold increase in the base composition of Sertoli cell RNA per mg DNA was observed over the age span of 18-35 days, with no preferential increase in any one specific nucleotide. There was no change in [3H]uridine incorporation (2 h) into RNA per cell (pmol/mg DNA), but decreased specific activity of the RNA (pmol/mg RNA) in Sertoli cells cultured from 35-day-old rats as compared to those from 18- to 19-day-old rats. Similar differences were noted in the specific activity of label incorporated into specific RNA bases. In contrast, the specific activity of the UTP-CTP soluble pool/mg DNA was only slightly increased. These data indicate that processes related to RNA synthesis in the Sertoli cell undergo a number of changes during the period of sexual maturation.     

Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg²⁺ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V _max of 360 pkat under optimal reaction conditions declining to v _limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K _m APS: 2 · 10^-6 mol · l^-1, and K _m ATP: 7 · 10^-6 mol · l^-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS Adenosine 5-phosphosulphate - dATP 2-deoxyadenosine 5-triphosphate - p-CMB p-chloromercuribenzoate - DTE dithioerythritol - DTT dithiothreitol - -MSH -mercaptoethanol - PAPS 3-phosphoadenosine 5-phosphosulphate - PAP 3-phosphoadenosine 5-phosphate - SDS sodium dodecyl sulphate This work is part of a dissertation submitted by H. G. J., Bochum 1982     

Tunicamycin inhibits prostaglandin F2α receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes

Effect of tunicamycin, an inhibitor of N-linked glycosylation, on prostaglandin (PG) F₂-stimulated phosphoinositide (PI) hydrolysis was examined in cultured rat astrocytes. Pretreatment of cultured astrocytes with tunicamycin (25–250 ng/ml) inhibited subsequent PGF₂ (1 M)-stimulated PI hydrolysis in concentration- and time-dependent manners. The inhibition completely recovered after removal of tunicamycin and re-incubation for 12 h. Tunicamycin pretreatment (100 ng/ml for 12 h) significantly blocked [³⁵S]methionine incorporation into cultured astrocytes, but cell viability was not affected under the condition. Inhibitors of processing of N-linked sugar chains such as bromoconduritol, 1-deoxymannojirimycin, and swainsonine had no effect on PI response to PGF₂. These observations suggest that PGF₂ receptor is N-linked glycosylated.     

Adenine nucleotide metabolism in Azotobacter vinelandii. Two metabolic pathways of AMP degradation

AMP-degrading pathways in Azotobacter vinelandii cells were investigated. AMP nucleosidase (EC 3.2.2.4) was rapidly synthesized and reached a maximum at 24 h, while the activity of 5-nucleotidase (EC 3.1.3.5) specific for AMP, which was negligible during the logarithmic phase of the growth, first appeared in 24 h-cultures, and reached a maximum after complete exhaustion of sucrose from the growth medium (70 h).Cell-free extracts of A. vinelandii of 48 h-cultures hydrolyzed AMP to ribose 5-phosphate and adenine in the presence of ATP, and adenine was deaminated to hypoxanthine. When ATP was excluded, AMP was dephosphorylated to adenosine, which was further metabolized to inosine, and finally to hypoxanthine. Hypoxanthine thus formed was reutilized for the salvage synthesis of IMP under the conditions where 5-phosphoribosyl 1-pyrophosphate was able to be supplied. These results suggest that the levels of ATP can determine the rate of AMP degradation by the AMP nucleosidase- and 5-nucleotidase-pathways. The role of ATP in the AMP degradation was discussed in relation to the regulatory properties of AMP nucleosidase, inosine nucleosidase (EC 3.2.2.2) and adenosine deaminase (EC 3.5.4.4).     

Formation of nucleoside 5′-phosphoramidates under potentially prebiological conditions

Summary Adenosine 5-phosphoramidates form when solutions containing adenosine 5-polyphosphates p_nA (n 3) or P₁, P₂-diadenosine 5-diphosphate and amines are allowed to dry out. Mg ions catalyze these reactions. We have studied systems containing ammonia, imidazole, glycine, ethylenediamine and histamine. The yields of adenosine 5-phosphoramidates range from 10–50 % based on the nucleotide. The prebiotic significance of the reactions is discussed.Abbreviations Im imidazole - hist histamine - gly glycine - en ethylenediamine - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - P_n (n = 1, 2 ) linear polyphosphate containing n phosphate residues - p_nA adenosine 5-polyphosphate containing n phosphate residues - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - AppA P₁, P₂-diadenosine 5-diphosphate - gly-pA adenylyl-(5N)-glycine - ImpA adenosine 5-phosphorimidazolide - NH₂-pA adenosine 5-phosphoramidate - en-pA adenylyl-(5N)-ethylenediamine - hist (NH) - pA adenosine 5-phospho-[2-(4-imidazolyl)-ethylamide] - hist(Im)-pA adenosine 5-phospho-[4-(2-aminoethyl)-imidazolide] - enP_1,2 phosphoramidates of ethylenediamine derived from H₃PO₄ and H₄P₂O₇     

ATP-dependent activation of phospholipase C by antigen,NECA, Na3VO4, and GTP-γ-S in permeabilized RBL cell ghosts: Differential augmentation by ATP,phosphoenolpyruvate and phosphocreatine

Ghosts prepared from rat basophilic leukemia cells (RBL cell ghosts) and permeabilized with -toxin fromS. aureus are a simplified system for the study of FcRI-mediated activation of phospholipase C (PLC). This activity is dependent upon ATP and magnesium, and is enhanced by the addition of another compound containing an energetic phosphate group, either phosphoenolpyruvate (PEP) or phosphocreatine (PCr). This effect appears to be specific for PEP and PCr in that other compounds with energetic phosphate bonds including fructose 1,6-bisphosphate and additional ATP are not effective. On the contrary, GTP--S, an activator of G proteins, activates PLC in the presence of ATP alone and this is not further enhanced by the addition of PEP. In addition to FcRI and GTP--S, two other stimuli lead to enhanced activity of PLC in permeabilized RBL cell ghosts: 1) an inhibitor of tyrosine phosphatases (Na₃VO₄) and 2) an analog of adenosine (NECA). Data presented here extend previous results to show that activation of PLC by GTP--S is not enhanced either by the addition of PCr or by the addition of a more MgATP. Further new findings include the observations that activation of PLC by Na₃VO₄ is augmented by PEP and PCr in a fashion similar to that observed for FcRI-mediated activation of PLC and that activation of PLC by NECA shows even more marked dependency on PEP than does activation by FcRI or Na₃VO₄. Together, these experiments demonstrate that antigen, Na₃VO₄, GTP--S, and NECA can all activate PLC in permeabilized RBL cell ghosts in the presence of ATP and that these activities are differentially affected by the addition of a second compound with an energetic phosphate bond.Abbreviations DNP₂₅BSA bovine serum albumin derivitized with 25 dinitrophenyl groups per mole of BSA - FcRI high affinity receptor for IgE - GTP--S guanosine-5-O-(3-thiotriphosphate) - IPs inositol phosphate(s) - K₂ Pipes dipotassium Pipes - Na₃VO₄ sodium vanadate - NECA 5-(N-Ethyl)carboxamidoadenosine - PCr phosphocreatine - PEP phosphoenolpyruvate - PLC phospholipase C specific for phosphoinositides - RBL rat basophilic leukemia     

Selective Cytostatic and Neurotoxic Effects of Avermectins and Activation of the GABAα Receptors

A natural avermectin complex, aversectin C, was shown to be capable of exerting selective cytostatic and neurotoxic effects on mammalian cells. Specifically, it killed proliferating neuroblastoma B103 cells but was non-toxic for differentiated cells of this culture. The anti-proliferation action of aversectin C was not inhibited by bicuculline or picrotoxin, antagonists of the GABA receptors, and was partly due to the action of avermectin A₁, a component of aversectin C. Aversectin C irreversibly suppressed activity of 60% neurons in medial septal slices of the rat brain. More than 55% of them were the GABA- and B₁-sensitive neurons whereas the rest, about 45% neurons, were the GABA-insensitive and the neurotoxic effect of aversectin C was caused mainly by the B₂ component.     

Desensitization of prostaglandin F2α receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes

Desensitization of prostaglandin (PG) F₂ receptor-mediated phosphoinositide (PI) hydrolysis was investigated in cultured rat astrocytes. Prolonged exposure of astrocytes differentiated by dibutyryl cyclic AMP-treatment to PGF₂ caused the desensitization of subsequent PGF₂-induced PI hydrolysis. The desensitization was time- and PGF₂ dose-dependent; maximal decrease in the PI hydrolysis was observed after exposure to 10 M PGF₂ for 4 h and the degree of the desensitization was 31.7±2.7% of control. Pretreatment with either PGD₂ or PGE₂ also induced the desensitization of subsequent PGF₂-stimulated PI hydrolysis and conversely pretreatment of PGF₂ decreased the PI responses to PGD₂ and PGE₂. The desensitization prevented by phloretin and was reversible upon removal of the agonist. Protein synthesis inhibitors blocked the recovery of the desensitization. Treatment of the cells with phorbol 12-myristate 13-acetate had no effect on the desensitization. These results suggest that prolonged exposure of the astrocytes to PGF₂ caused the desensitization of the receptors.     

New sialyltransferase inhibitors based on CMP-quinic acid: development of a new sialyltransferase assay

Quinic acid (4) was transformed into phosphitamides 6, 14, and 15, which could be readily linked to 5-O-unprotected cytidine derivative 7; ensuing oxidation of the obtained phosphite triesters with tert-butylhydroperoxide furnished the corresponding phosphate triesters 8, 16, and 17, respectively. Hydrogenolytic debenzylation of the phosphate moiety, base catalysed removal of acetyl protective groups, and basic hydrolysis of the methylester of the quinic acid moiety furnished CMP-Neu5Ac analogues 1-3. In order to measure their inhibition of sialyltransferases, a nonradioactive sialyltransferase assay [employed for (2-6)-sialyltransferase from rat liver (EC 2.4.99.1)] based on reversed-phase HPLC separation of UV-abelled acceptor 20 (p-nitrophenyl glycoside of N-acetyllactosamine) from the UV-labelled product 21 (p-nitrophenyl glycoside of sialyl (2-6)-N-acetyllactosamine) and p-nitrophenylalanine as internal standard was developed. The assay reproduced the reported KM values for CMP-Neu5Ac and N-acetyllactosamine and the Ki values for CDP. 1 and 2 turned out to be potent sialyltransferase inhibitors. © 1998 Rapid Science Ltd     

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells

Localisation of -amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of -amylase protein from aleurone cells.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - PBS phosphate-buffered saline     

In vitro effects of thyroid hormones on ectonucleotidase activities in synaptosomes from hippocampus of rats

1. Studies have shown that adenosine transport and adenosine A1 receptors in rat brain are subjected to regulation by thyroid hormone levels. Since the ectonucleotidase pathway is an important source of adenosine extracellular, in the present study the in vitro action of T3 and T4 hormones on ectonucleotidase activities in hippocampal synaptosomes was evaluated.2. T3 (Triiodo-l-thyronine) significantly inhibited, in an uncompetitive manner, the ATP and ADP hydrolysis promoted by ATP diphosphohydrolase activity in hippocampal synaptosomes of adult rats.3. In contrast, T4 (Thyroxine) only inhibited ATP hydrolysis in an uncompetitive mechanism, at the concentrations tested (100–500 M), but at the same time did not affect ADP hydrolysis.4. In the present study, we also investigate the in vitro effect of T3 and T4 on 5-nucleotidase activity. However, there are no changes in the activity of this enzyme in the presence of T3 and T4 in the hippocampal synaptosomes of rats.5. These results suggest that thyroid hormones could be involved in the regulation of ectonucleotidase activities, such as ecto-ATP diphosphohydrolase and ecto-ATPase, possibly exerting a modulatory role in extracellular adenosine levels.     

DNA Polymerase-Associated Lectin (DPAL) and Its Binding to the Galactose-Containing Glycoconjugate of the Replication Complex

The highly purified DNA Pol- from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488–1492]. Binding of ConA and RCA to human recombinant DNA polymerase- showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231–6237].The catalytic polypeptide, DNA polymerase- of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase- from embryonic chicken brain (ECB) contains an -galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase- reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567–573] by the treatment with methyl--galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (M _r = 186 kDa) with an -galactose binding, 56 kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA.Pol- as determined by immunostaining with the polymerase--specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA- and a complete separation of polymerase complex and primase.     

Isolation and characterization of the mitochondrial ATP synthase fromChlamydomonas reinhardtii. cDNA sequence and deduced protein sequence of the α subunit

We have isolated the F₀F₁-ATP synthase complex from oligomycin-sensitive mitochondria of the green algaChlamydomonas reinhardtii. A pure and active ATP synthase was obtained by eans of sonication, extraction with dodecyl maltoside and ion exchange and gel permeation chromatography in the presence of glycerol, DTT, ATP and-21. The enzyme consists of 14 subunits as judged by SDS-PAGE. A cDNA clone encoding the ATP synthase subunit has been sequenced. The deduced protein sequence contains a presequence of 45 amino acids which is not present in the mature protein. The mature protein is 58–70% identical to corresponding mitochondrial proteins from other organisms. In contrast to the ATP synthase subunit fromC. reinhardtii (Franzen and Falk, Plant Mol Biol 19 (1992) 771–780), the protein does not have a C-terminal extension. However, the N-terminal domain of the mature protein is 15–18 residues longer than in ATP synthase subunits from other organisms. Southern blot analysis indicates that the protein is encoded by a single-copy gene.Abbreviations DM dodecyl--D-maltoside - OSCP oligomycin sensitivity conferring protein - PMSF phenyl-methylsulfonylfluoride - DTT dithiothreitol - EDTA ethylenediaminotetraacetic disodium salt     

Partial purification and characterization of alpha-glucosidase from Pseudomonas fluorescens W

The -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.20) of Pseudomonas fluorescens W was partially purified by (NH₄)₂SO₄ fractionation, Sephadex G-200 and DEAE-cellulose column chromatography. The enzyme showed great specificity for maltose hydrolysis, with very little action against polymeric forms. Sucrose, isomaltose, -methylglucoside, and maltobionic acid were not hydrolyzed. Turanose was a strong competitive inhibitor, and glucose a weaker one. Tris (2-amino-2-hydroxymethylpropan-1:3-diol) inhibited enzyme activity significantly only at alkaline pH. Mercuric, cupric, and silver cations strongly inhibited, and EDTA (ethylenediaminetetraacetate) weakly inhibited the enzyme. The isolated enzyme was rather unstable even at 4° C, and was destroyed by freezing and lyophilization. Inositol and albumin had a slightly protective effect. Sulfhydryl-binding reagents strongly inhibited the enzyme.Abbreviations PNPG paranitrophenyl--d-glucoside - PCMB parachloromercuribenzoate - DEAE diethylaminoethyl cellulose - NEM N-ethylmaleimide - EDTA ethylenediaminetetraacetate     

Transport and posttranslational processing of the vacuolar enzyme α-mannosidase in jack-bean cotyledons

-Mannosidase (EC 3.2.1.24) is a vacuolar enzyme which occurs abundantly in the cotyledons of the jack-bean (Canavalia ensiformis (L.) DC). The mature enzyme is a tetramer with two polypeptides each of relative molecular mass (M_r) 66000 and M_r 44000. The enzyme has an interesting molecular structure because in its native form, it does not bind to concanavalin A (ConA) in spite of the presence of a high-mannose glycan. -Mannosidase is synthesized in the developing cotyledons of jack-beans at the same time as the abundant proteins canavalin and ConA. The enzyme is synthesized as a precursor which has an M_r of 110000 and is associated with the endoplasmic reticulum (ER). Antibodies against the deglycosylated subunits cross-react with the M_r-110000 precursor. Processing of the precursor to the constituent polypeptides occurs posttranslationally, probably in the protein bodies. Immunocytochemical evidence shows that -mannosidase is present in the ER and the Golgi complex of developing cells, and accumulates in the protein bodies.Labeling with [³H]glucosamine shows that after processing only the M_r-66000 polypeptide has glucosamine-containing glycans. The synthesis of these glycans is inhibited by tunicamycin, indicating that they are asparagine-linked oligosaccharides. Analysis of the glycans shows that there is a large glycan that is retained by ConA and a small glycan that is not retained by ConA. The large glycan is only partially sensitive to -mannosidase because of the presence of a terminal glucose residue. Cross-reaction of the large subunit with an antiserum directed against small, complex glycans of plant glycoproteins indicates that this polypeptide probably has a xylose-containing glycan. Pulse-chase experiments carried out in the presence of tunicamycin show that the presence of glycans is not required for transport of -mannosidase out of the ER-Golgi system.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - H L heavy, light subunit - IgG Immunoglobulin G - M_r relative molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Molecular cloning,sequencing and expression of an α2-adrenergic receptor complementary DNA from rat brain

We have isolated a cDNA clone from rat brain using a human platelet ₂-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney ₂-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5-untranslated region. Southern-blot analysis with a human platelet ₂-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain ₂-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A⁺) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the ₂-adrenergic antagonist [³H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the _2A subclass. We conclude that the rat brain cDNA encodes a new ₂-adrenergic receptor subtype that may be brain-specific.Abbreviations G protein guanine nucleotide-binding proteins - cA2-47 ₂-adrenergic receptor cDNA from rat brain - SSC (1X SSC contains 0.15 M NaCl, 15 mM Na₃citrate, pH 7.0)     

The paracrine role of Sertoli cells on Leydig cell function

Conclusion Local regulation of testicular function depends upon multiple interactions between testicularcells, some of them mediated by soluble factors (Fig. 2). Under physiological conditionsgonadotropins are required for testicular maturation and function, but the responsiveness ofsomatic testicular cells to these hormones is modulated by factors produced and acting withinthe testis. Moreover, the production of these factors, and sometimes the responsiveness oftesticular cells to these factors, are gonadotropin-dependent. While the paracrine regulation ofSertoli function by Leydig cells seems to be mediated mainly by a direct or indirect effects oftestosterone, several factors seem to be involved in the FSH-stimulated effects of Sertoli cellson Leydig cells. The nature of the factors implicated in Sertoli-germ cell interactions ispractically unknown. The amounts of these factors might be very low, although sufficient toact in a paracrine, autocrine, juxtracrine or cryptocrine manner, and their isolation andpurification would be a very difficult task.Abbreviations EGF: epidermal growth factor - FSH: follicle-stimulating hormone - GH: growth hormone - hCG: human chorionic gonadotropin - IGF-I: insulin-like growth factor I - LH: luteinizing hormone - TGF-: transforming growth factor - TGF-: transforming growth factor      

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of A- and B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to B-crystallin, but not to A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to A- and B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to A- and B-crystallin. In rat embryos, A-crystallin appeared in the lens pit at E12, and B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to A-crystallin. The lens fiber cells were also immunoreactive to B-crystallin, but the epithelial cells were not. These findings suggest that B-crystallin appears earlier than A-crystallin in the human lens, but at a later period than A-crystallin in the rat lens. B-Crystallin was not detected in the epithelial cells of the rat lens, but was perisistently present in the epithelial cells of the human lens.