Myoinositol biosynthesis by Sertoli cells, and levels of myoinositol biosynthetic enzymes in testis and epididymis

1979 ◽  
Vol 57 (6) ◽  
pp. 962-967 ◽  
Author(s):  
Ranga Robinson ◽  
Irving B. Fritz
Keyword(s):  
Sertoli Cells ◽  
Specific Activity ◽  
Primary Cultures ◽  
Defined Medium ◽  
Seminiferous Tubules ◽  
Chemically Defined Medium ◽  
Dibutyryl Cyclic Amp ◽  
Old Rats ◽  
Specific Activities ◽  
Germinal Cells

Levels of glucose-6-phosphate cyclase (myoinositol-1-phosphate synthase, EC 5.5.1.4) and myoinositol-1-phosphate phosphatase (myoinositol-1-phosphatase, EC 3.1.3.25) were determined in extracts of testes from 10-, 20-, and 30-day-old rats, and in extracts of Sertoli cells, germinal cells, and epididymides. The specific activity of the cyclase was approximately [Formula: see text] that of the phosphatase in all extracts found to contain either enzyme. Among cells in the testis examined, Sertoli cells had highest levels of enzymes required for inositol biosynthesis from glucose, while spermatocytes and round spermatids did not have detectable activity. Spermatozoa from the epididymis also had no detectable cyclase or phosphatase activity. In contrast, extracts of washed epididymides contained exceedingly high specific activities of these enzymes. Primary cultures of Sertoli cells, maintained in a chemically defined medium without added inositol, released inositol into the medium during three successive 24-h periods. The amounts released were greater in cells stimulated by dibutyryl cyclic AMP. Results were interpreted to indicate that inositol in the fluid of seminiferous tubules most probably originates from Sertoli cells, which synthesize inositol from glucose. Additional inositol in the fluid of epididymal tubules could readily be provided by metabolism of glucose by epididymal epithelial cells.

scholarly journals Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis

2006 ◽  
Vol 190 (1) ◽  
pp. 59-71 ◽  
Author(s):  
Sophie Fouchécourt ◽  
Murielle Godet ◽  
Odile Sabido ◽  
Philippe Durand
Keyword(s):  
Cell Line ◽  
Glial Cell ◽  
Sertoli Cells ◽  
Small Population ◽  
Seminiferous Tubules ◽  
Rat Testis ◽  
Old Rats ◽  
Neurotropic Factor ◽  
Pachytene Spermatocytes ◽  
Factor Α

Glial cell-line-derived neurotropic factor (GDNF) and its receptors glial cell-line-derived neurotropic factor α (GFR1α) and rearranged during transformation (RET) have been localized in the rat testis during postnatal development. The three mRNAs, and GDNF and GFR1α proteins were detected in testis extracts from 1- to 90-day-old rats by reverse transcriptase PCR and Western blotting respectively. The three mRNAs were present in Sertoli cells from 20- and 55-day-old rats, pachytene spermatocytes (PS), and round spermatids (RS). The GDNF and GFR1α proteins were detected in PS, RS, and Sertoli cells. GDNF and GFR1α were also detected using flow cytometry in spermatogonia and preleptotene spermatocytes, and in secondary spermatocytes. The localization of GDNF and GFR1α in germ and Sertoli cells was confirmed by immunocytochemistry. The hypothesis that GDNF may control DNA synthesis of Sertoli cells and/or spermatogonia in the immature rat was addressed using cultures of seminiferous tubules from 7- to 8-day-old rats. Addition of GDNF for 48 h resulted in a twofold decrease in the percentage of spermatogonia able to duplicate DNA, whereas Sertoli cells were not affected. These results are consistent with a role of GDNF in inhibiting the S-phase entrance of a large subset of differentiated type A spermatogonia, together with an enhancing effect of the factor on a small population of undifferentiated (stem cells) spermatogonia. Moreover, the wide temporal and spatial expression of GDNF and its receptors in the rat testis suggest that it might act at several stages of spermatogenesis.

Nitrogen source regulates expression of alanine dehydrogenase isoenzymes in Streptomyces avermitilis in a chemically defined medium

1997 ◽  
Vol 43 (2) ◽  
pp. 189-193 ◽  
Author(s):  
Jan Novák ◽  
Jan Kopecký ◽  
Zdenko Vaněk
Keyword(s):  
Nitrogen Source ◽  
Ammonium Sulfate ◽  
Alanine Aminotransferase ◽  
Specific Activity ◽  
Defined Medium ◽  
Streptomyces Avermitilis ◽  
Chemically Defined Medium ◽  
Ammonium Ions ◽  
Alanine Dehydrogenase ◽  
Dehydrogenase Reaction

Ammonium ions and alanine influence production of the macrolide avermectin in Streptomyces avermitilis. L-Alanine dehydrogenase and alanine aminotransferase are the primary enzymes responsible for regulating the intracellular concentration of alanine and also of ammonium ions. In cultures of S. avermitilis in a chemically defined medium with ammonia or L-alanine as the only nitrogen source, specific activities of both enzymes increased during growth. The alanine dehydrogenase specific activity increased more than 86-fold after the culture was supplemented with 0.2% L-alanine and 5-fold after addition of 0.5% ammonium sulfate, whereas alanine aminotransferase specific activity increased 3- to 4-fold with either substrate. Five isoenzymes of alanine dehydrogenase were detected histochemically in S. avermitilis after native gel electrophoresis. Isoenzyme 1 was induced by alanine and temporarily repressed by high concentrations of ammonium sulfate. The presence of isoenzyme 1 was also related to changes in the kinetic properties of the alanine dehydrogenase reaction measured in crude desalted extracts. A nonlinear double-reciprocal plot was obtained in initial velocity studies using L-alanine as a substrate in the sample induced with L-alanine. The nonlinearity was caused by both substrate inhibition and allosteric regulation (positive cooperativity) by L-alanine. In contrast, the sample induced by ammonium sulfate showed a linear double-reciprocal plot.Key words: isoenzymes, L-alanine dehydrogenase, Streptomyces avermitilis, avermectin.

scholarly journals Age-Related Changes in Hepatic Activity and Expression of Detoxification Enzymes in Male Rats

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Erika Vyskočilová ◽  
Barbora Szotáková ◽  
Lenka Skálová ◽  
Hana Bártíková ◽  
Jitka Hlaváčová ◽  
...  
Keyword(s):  
Antioxidant Enzymes ◽  
Specific Activity ◽  
Detoxification Enzymes ◽  
Male Rats ◽  
Cellular Functions ◽  
Metabolizing Enzymes ◽  
Age Related ◽  
Male Wistar Rats ◽  
Old Rats ◽  
Specific Activities

Process of aging is accompanied by changes in the biotransformation of xenobiotics and impairment of normal cellular functions by free radicals. Therefore, this study was designed to determine age-related differences in the activities and/or expressions of selected drug-metabolizing and antioxidant enzymes in young and old rats. Specific activities of 8 drug-metabolizing enzymes and 4 antioxidant enzymes were assessed in hepatic subcellular fractions of 6-week-old and 21-month-old male Wistar rats. Protein expressions of carbonyl reductase 1 (CBR1) and glutathioneS-transferase (GST) were determined using immunoblotting. Remarkable age-related decrease in specific activities of CYP2B, CYP3A, and UDP-glucuronosyl transferase was observed, whereas no changes in activities of CYP1A2, flavine monooxygenase, aldo-keto reductase 1C, and antioxidant enzymes with advancing age were found. On the other hand, specific activity of CBR1 and GST was 2.4 folds and 5.6 folds higher in the senescent rats compared with the young ones, respectively. Interindividual variability in CBR1 activity increased significantly with rising age. We suppose that elevated activities of GST and CBR1 may protect senescent rats against xenobiotic as well as eobiotic electrophiles and reactive carbonyls, but they may alter metabolism of drugs, which are CBR1 and especially GSTs substrates.

scholarly journals GROWTH CHARACTERISTICS OF MONKEY KIDNEY CELL STRAINS LLC-MK1, LLC-MK2, AND LLC-MK2(NCTC-3196) AND THEIR UTILITY IN VIRUS RESEARCH

1962 ◽  
Vol 115 (5) ◽  
pp. 903-918 ◽  
Author(s):  
R. N. Hull ◽  
W. R. Cherry ◽  
O. J. Tritch
Keyword(s):  
Primary Cultures ◽  
Defined Medium ◽  
Growth Characteristics ◽  
Monkey Kidney ◽  
Freeze Storage ◽  
Kidney Cells ◽  
Chemically Defined Medium ◽  
Monkey Kidney Cell ◽  
Virus Research ◽  
Cell Strains

The establishment of two strains of rhesus monkey kidney cells in continuous tissue culture, the development of a subline adapted to chemically defined medium, and the isolation of several clonal derivatives were described. Growth characteristics, chromosome numbers, malignant potentiality, and freeze storage data are presented. The cells were studied for their sensitivity to a large number of viruses and were extensively compared with primary cultures of monkey kidney cells for sensitivity to poliovirus. The cell strains were not sensitive to all the viruses which could be grown in primary cultures of the same tissue but were susceptible to most of them. In some instances an advantage to the use of the cell strain for certain viruses was noted.

scholarly journals Tryptophan catabolism during sporulation in Bacillus cereus

1970 ◽  
Vol 119 (2) ◽  
pp. 343-349 ◽  
Author(s):  
Chandan Prasad ◽  
V. R. Srinivasan
Keyword(s):  
Bacillus Cereus ◽  
Anthranilic Acid ◽  
Maximal Activity ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Whole Cells ◽  
Stage 4 ◽  
Tryptophan Catabolism ◽  
Tryptophan Pyrrolase ◽  
Specific Activities

1. Two intermediates of tryptophan catabolism were isolated from a sporulating culture of Bacillus cereus and identified as anthranilic acid and kynurenine by their spectral properties. 2. During sporulation the rate of formation of anthranilic acid and kynurenine by whole cells increased and reached a maximum at the pre-spore stage. 3. The specific activities of tryptophan pyrrolase and formylase also increased during sporulation and exhibited a maximal activity at the pre-spore stage. 4. Kynureninase activity reached a maximum during early stages of sporulation and then started to decline. 5. There was a net increase in the activity of tryptophan pyrrolase when cells were grown in the presence of l-tryptophan or dl-kynurenine. 6. The cultures exhibited the maximal activity of kynureninase 2h earlier in the presence of dl-kynurenine whereas l-tryptophan delayed the appearance of the maximal activity by 2h. 7. The omission of glucose from the medium had no effect on the pattern of development of tryptophan pyrrolase during growth and sporulation. 8. On the addition of tryptophan to a chemically defined medium no significant change in the pattern of development of tryptophan pyrrolase was observed.

METABOLIC CHANGES IN TESTICULAR CELLS FROM RATS AFTER LONG-TERM EXPOSURE TO 37 °C IN VIVO OR IN VITRO

1980 ◽  
Vol 85 (3) ◽  
pp. 471-479 ◽  
Author(s):  
F. F. G. ROMMERTS ◽  
F. H. DE JONG ◽  
J. A. GROOTEGOED ◽  
H. J. VAN DER MOLEN
Keyword(s):  
Sertoli Cells ◽  
Leydig Cells ◽  
Biochemical Properties ◽  
Effect Of Temperature ◽  
Seminiferous Tubules ◽  
Experimental Conditions ◽  
Testicular Cells ◽  
Old Rats

Biochemical properties of isolated Leydig cells, Sertoli cells and spermatocytes from rat testes have been investigated after in-vivo or in-vitro exposure of these cells to abdominal temperature (37 °C). The rate of production of testosterone and pregnenolone by isolated Leydig cells from cryptorchid and normal testes from mature rats was not different. Production of pregnenolone by mitochondria prepared from cryptorchid testes was 6·7 times higher than production by mitochondria from normal testes. Sertoli cells prepared from immature rats and incubated in vitro at 32 or 37 °C showed, on day 1 of the culture period, an initial twofold increase in the secretion of androgen-binding protein which was absent after 6 days in culture. In contrast, incorporation of [3H]leucine into secreted proteins was stimulated twofold on day 1 as well as by day 6 of culture. Secretion of oestradiol was increased 30-fold by day 6 when compared with the level found on day 1 when cells had been cultured at 37 °C and the increased secretion of oestradiol was maintained for approximately 2 days when the temperature of incubation was decreased to 32 °C Spermatocytes isolated from seminiferous tubules incubated for 20 h at 37 °C were active in the synthesis of RNA. No degeneration of these cells was observed in testes of 25-day-old rats 5 days after experimental cryptorchidism, whereas under similar conditions massive degeneration of spermatocytes was shown in the testes of mature rats. These results suggest that the effects of temperature on the different testicular cells greatly depend on the experimental conditions used to study the effect of temperature.

241 DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS

2009 ◽  
Vol 21 (1) ◽  
pp. 218
Author(s):  
Y. Akaki ◽  
K. Yoshioka ◽  
H. Funahashi
Keyword(s):  
Cyclic Amp ◽  
In Vitro Maturation ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Chemically Defined Medium ◽  
Dibutyryl Cyclic Amp ◽  
Vitro Fertilization ◽  
Porcine Oocyte

Exposure of porcine oocyte–cumulus complexes (OCC) to gonadotropins induces meiotic resumption, but the details of this mechanism are still unknown. The present study was undertaken to examine combinational effects of EGF-like factors and dibutyryl cyclic AMP (dbcAMP) in a chemically defined medium on in vitro maturation (IVM) of porcine oocytes. The OCC were aspirated from 3- to 6-mm-diameter follicles of prepuberal ovaries and used in the current study. The basic culture medium was a chemically defined medium, Porcine Oocyte Medium (POM; Research Institute for the Functional Peptides, Yamagata, Japan). In the first experiment, various concentrations (0, 10, and 1000 ng mL–1) of EGF-like factors (EGF, amphiregulin, and betacellulin) were added to POM during an entire IVM period (44 h). In the second experiment, to determine the additive effect of EGF-like factors, each EGF-like factor with an effective concentration was combined with the others. In the last experiment, to examine the combined effect with dbcAMP, OCC were exposed to EGF (10 ng mL–1), amphiregulin (1000 ng mL–1), and dbcAMP (1 mm) during the first 20 h of IVM and then the culture was continued in the absence of EGF-like factors and dbcAMP. After culture, in all experiments, meiotic resumption and the progress of oocytes were examined after denuding, fixing, and staining. Statistical analyses was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In the first experiment, all treatments without supplementation with 10 ng mL–1 amphiregulin increased the incidence of oocytes maturing to the MII phase, as compared with controls (29.1 to 39.3% v. 11.1%, P < 0.05). In the second experiment, combinations with 2 kinds of EGF-like factor slightly (but not significantly) improved the percentage of oocytes at the MII stage (37.7 to 47.4%). In the last experiment, supplementation with 1 mm dbcAMP during the first 20 h of IVM, regardless of the presence of EGF-like factors, significantly increased the incidence of MII oocytes as compared with controls, whereas the incidence was the highest when 1 mm dbcAMP, 10 ng mL–1 EGF, and 1000 ng mL–1 amphiregulin were supplemented (75.5%). When those oocytes were cultured in a chemically defined medium after in vitro fertilization, the developmental competence of oocytes to the blastocyst stage (25.0%) was not different from oocytes matured in the presence of gonadotropins and dbcAMP during the first 20 h of IVM (17.3%). These observations indicate that supplementation of a chemically defined maturation medium with EGF-like factors and dbcAMP during the first 20 h of IVM can support the meiotic progress and developmental competence of porcine oocytes well. Currently, we are examining the developmental competence of those oocytes after embryo transfer. The results will be presented at the meeting. This study was supported by MAFF AgriBio1605.

Regulation of stages VI and VIII of the rat seminiferous epithelial cycle in vitro

1986 ◽  
Vol 108 (3) ◽  
pp. 417-NP ◽  
Author(s):  
J. Toppari ◽  
K. K. Vihko ◽  
K. G. E. Räsänen ◽  
E. Eerola ◽  
M. Parvinen
Keyword(s):  
Retinoic Acid ◽  
Follicle Stimulating Hormone ◽  
Defined Medium ◽  
Seminiferous Tubules ◽  
Chemically Defined Medium ◽  
Flow Cytometric ◽  
Culture Stage ◽  
Normal Differentiation ◽  
Stage Viii

ABSTRACT Plasminogen activator (PA) is secreted cyclically (in stages VII and VIII) by rat seminiferous tubules. To investigate whether this can be maintained and influenced in vitro, tubule segments from stages VI and VIII of the epithelial cycle were cultured for 3 days in chemically defined medium supplemented with testosterone, FSH, or a combination of testosterone, FSH, insulin and retinoic acid (4F). Morphological and flow cytometric analyses of stage VI tubules suggested a roughly normal differentiation to stage VIII. They developed an increased PA secretion on day 3 of culture. Stage VIII tubules, however, did not develop all the characteristics of stage XII. Step 8 spermatids did not elongate and step 19 spermatids failed to develop into spermatozoa. Secretion of PA on day 3 was not significantly different to that on day 1. The 4F combination very significantly stimulated PA secretion in both stages, but FSH alone was effective only in stage VIII. Most of the secreted PA had a molecular weight of 43 000 in both stages, suggesting that it is of urokinase type. The results suggest that stage VI is more able to differentiate in vitro for 3 days than stage VIII; the cyclic secretion pattern of PA was partially maintained in tubule segments from stage VI. Follicle-stimulating hormone had an effect on PA secretion only in stage VIII, whereas the 4F combination was stimulatory in both stages. The retinoic acid in this combination may be of importance in the regulation of PA secretion by seminiferous tubules. J. Endocr. (1986) 108, 417–422

INFLUENCE OF GERM CELLS UPON TRANSFERRIN SECRETION BY RAT SERTOLI CELLS in vitro

1988 ◽  
Vol 118 (3) ◽  
pp. R13-R16 ◽  
Author(s):  
B. LE MAGUERESSE ◽  
C. PINEAU ◽  
F. GUILLOU ◽  
B. JEGOU
Keyword(s):  
Sertoli Cell ◽  
Cell Cultures ◽  
Germ Cells ◽  
Sertoli Cells ◽  
Seminiferous Tubules ◽  
Adult Rats ◽  
Hypotonic Treatment ◽  
Old Rats ◽  
Pachytene Spermatocytes

ABSTRACT Indirect approach (hypotonic treatment) and direct approaches (co-cultures and conditioned media) were used in order to investigate the effects of germ cells from adult rats upon transferrin secretion by Sertoli cell cultures prepared from 20-day-old rats. Removal of germ cells contaminating the Sertoli cell cultures resulted in a significant decrease in transferrin secretion whereas the addition of crude germ cell preparations or of enriched preparations of pachytene spermatocytes, early spermatids and of liver epithelial cells (LEC) markedly stimulated this parameter. Furthermore, spent media of pachytene spermatocytes and of early spermatids, but not of LEC, also stimulated transferrin production. It is concluded that germ cells normally located within the adluminal compartment of the seminiferous tubules may be capable of controlling their own supply of iron via their influence upon transferrin secretion by the Sertoli cells.

An International Collaborative Study to Investigate Standardisation of Hirudin Potency

1993 ◽  
Vol 69 (05) ◽  
pp. 430-435 ◽  
Author(s):  
Colin Longstaff ◽  
Man-Yu Wong ◽  
Patrick J Gaffney
Keyword(s):  
Specific Activity ◽  
Collaborative Study ◽  
Residual Activity ◽  
Quality Standard ◽  
Upper Limit ◽  
Assay Procedure ◽  
Specific Activities ◽  
International Collaborative ◽  
Range Of Values ◽  
International Collaborative Study

SummaryAn international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of α-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard α-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200–222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15–15.97%. This corresponds to specific activities of approximately 14,300–15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000–16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard α-thrombin by all laboratories. This study has important implications for hirudin standardisation.

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