IN-VITRO SYNTHESIS OF ANDROGEN FROM PREGNENOLONE IN THE TESTES OF THE GOAT (CAPRA HIRCUS) AND IDENTIFICATION OF 5-PREGNENE-3β,17α,20α-TRIOL AS AN INTERMEDIATE IN THE METABOLIC PATHWAY OF PREGNENOLONE

1980 ◽  
Vol 84 (3) ◽  
pp. 381-390 ◽  
Author(s):  
MAKOTO MORI ◽  
SACHIKO MATSUKURA ◽  
KAZUHIKO KAWAKURA ◽  
BUN-ICHI TAMAOKI
Keyword(s):  
Time Course ◽  
Microsomal Fraction ◽  
Testicular Tissue ◽  
The Other ◽  
Capra Hircus ◽  
Cytosol Fraction ◽  
In Vitro Synthesis ◽  
Intermediary Metabolite ◽  
End Products

When [4–14C]pregnenolone was aerobically incubated in vitro in the presence of NAD+ and NADPH with cell-free homogenates of testicular tissue of adult domestic goats (Capra hircus), progesterone, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, 17α,20α-dihydroxy-4-pregnen-3-one, dehydroepiandrosterone, androstenedione and testosterone were identified as its known metabolites. Time-course studies on this metabolism showed that the production of 17α,20α-dihydroxy-4-pregnen-3-one and testosterone constantly increased up to the end of incubation, suggesting that these are both end-products of pregnenolone metabolism in this system. The other metabolites behaved as intermediates and were ultimately converted, in part, to testosterone by the testicular homogenates, indicating that testosterone was synthesized through both 4-ene and 5-ene-pathways. Furthermore, besides these metabolites, 5-pregnene-3β,17α,20α-triol was also identified as an intermediary metabolite, formed from pregnenolone through 17α-hydroxypregnenolone in the presence of NADPH, and further convertible into 17α,20α-dihydroxy-4-pregnen-3-one by the microsomal fraction and into 17α-hydroxypregnenolone by the cytosol fraction in the presence of NAD+ and NADP It was not, however, significantly transformed into C19-steroids. Furthermore, 17α,20α-dihydroxy-4-pregnen-3-one, which was formed either from 5-pregnene-3β,17α,20α-triol or from 17α-hydroxyprogesterone, remained almost unchanged without conversion to C19-steroids when incubated with the caprine testicular homogenates.

BIOSYNTHESIS OF TESTOSTERONE AND OESTROGENS IN VITRO BY THE TESTICULAR TISSUE FROM PATIENTS WITH KLINEFELTER'S SYNDROME

1971 ◽  
Vol 66 (4) ◽  
pp. 737-744 ◽  
Author(s):  
Dinesh C. Sharma ◽  
J. Lester Gabrilove
Keyword(s):  
Normal Control ◽  
Testicular Tissue ◽  
Klinefelter’S Syndrome ◽  
Klinefelter's Syndrome ◽  
Positive Form ◽  
Control Subjects ◽  
End Products ◽  
Testis Tissue

ABSTRACT Testis tissue from patients with the chromatin positive form of Klinefelter's syndrome was incubated with 17α-hydroxyprogesterone and testosterone. The ratio of oestrogen to testosterone in the end products of the incubation utilizing 17α-hydroxyprogesterone as substrate was 5 to 10 times the ratio obtained in similar investigations employing testis from normal control subjects. An increased conversion into oestrogen of testosterone utilized as substrate was also observed in the in vitro studies of testis obtained from the patients with Klinefelter's syndrome. These data lend support to the thesis that in the chromatin positive form of Klinefelter's syndrome there is an increased conversion of testosterone into oestrogen in the testis.

Growth of the endometrium and cotyledons during pregnancy in the ewe: rates of protein secretion and synthesis and nuclear and cytosol steroid hormone receptor levels

1983 ◽  
Vol 96 (1) ◽  
pp. 137-146 ◽  
Author(s):  
B. G. Miller ◽  
R. Tassell ◽  
G. M. Stone
Keyword(s):  
Progesterone Receptor ◽  
Protein Secretion ◽  
Time Course ◽  
Steroid Hormone ◽  
Secretory Activity ◽  
The Other ◽  
Steroid Hormone Receptor ◽  
High Affinity ◽  
Nuclear Progesterone Receptor

The time-course of cell hypertrophy and changes in in-vitro rates of secretion and synthesis of protein in intercaruncular and caruncular endometrium and maternal and fetal cotyledonary placenta have been examined during days 0–112 of pregnancy in the ewe. The concentrations of high-affinity receptors for oestradiol and progesterone in nuclear and cytosol fractions from these tissues were also determined. Protein secretion by intercaruncular endometrium increased 25-fold between days 0 and 84. On day 84 10−5 m-colchicine blocked 75% of total secretion. Protein secretion did not increase in the other tissues. Protein synthesis and RNA: DNA ratio in intercaruncular endometrium increased steadily between days 0 and 112, whereas they did not change in caruncular endometrium between days 0 and 28 and declined in cotyledon between days 56 and 112. The levels of cytosol receptor for oestradiol and progesterone and of nuclear receptor for oestradiol in all tissues during days 56–112 were very low in relation to the corresponding levels in caruncular endometrium on day 0. The level of nuclear progesterone receptor in caruncular endometrium increased threefold between oestrus and day 28. The level of this receptor in cotyledon remained low on days 56–112, but in intercaruncular endometrium it increased to high values on days 84–112. The results demonstrated a major surge in secretory activity by the intercaruncular endometrium at around mid-gestation, which was associated with a marked increase in nuclear progesterone receptor levels but only a low level of nuclear oestradiol receptor. The observations do not suggest any important role for oestradiol or progesterone in the growth of fetal and maternal cotyledon.

scholarly journals Pathway of Glucose Catabolism by Strain VeGlc2, an Anaerobe Belonging to the Verrucomicrobiales Lineage of Bacterial Descent

1998 ◽  
Vol 64 (12) ◽  
pp. 4830-4833 ◽  
Author(s):  
Peter H. Janssen
Keyword(s):  
Electron Transport ◽  
Enzyme Activities ◽  
The Other ◽  
Difference Spectra ◽  
Glucose Catabolism ◽  
Cell Extracts ◽  
In Vitro Measurements ◽  
End Products ◽  
Reductive Pathway

ABSTRACT Strain VeGlc2, an anaerobic ultramicrobacterium belonging to theVerrucomicrobiales lineage of bacterial descent, fermented glucose to acetate, propionate, succinate, and CO2. The distribution of radiolabel in the fermentation end products produced from position-labelled glucose and in vitro measurements of enzyme activities in crude cell extracts prepared from glucose-grown cells showed that glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The 6-phosphofructokinase (EC 2.7.1.90 ) activity required pyrophosphate as the phosphoryl donor, and ATP could not replace pyrophosphate. The other enzyme activities were those of a classical Embden-Meyerhof-Parnas pathway. 14CO2 was incorporated into propionate and succinate, suggesting that a carboxylation reaction rather than a transcarboxylation reaction was involved in the reductive pathway leading to succinate and propionate. Difference spectra showed that a type b cytochrome was present, which could be involved in electron transport in the reductive pathway.

scholarly journals The separation of β-glucanases produced by Cytophaga johnsonii and their role in the lysis of yeast cell walls

1970 ◽  
Vol 120 (1) ◽  
pp. 67-78 ◽  
Author(s):  
J. S. D. Bacon ◽  
A. H. Gordon ◽  
D. Jones ◽  
Irene F. Tayor ◽  
D. M. Webley
Keyword(s):  
Yeast Cell ◽  
Cell Walls ◽  
Culture Fluid ◽  
The Other ◽  
Yeast Cells ◽  
Wall Structure ◽  
Enzyme Preparations ◽  
End Products ◽  
Isolated Cell

1. When Cytophaga johnsonii was grown in the presence of suitable inducers the culture fluid was capable of lysing thiol-treated yeast cell walls in vitro. 2. Autoclaved or alkali-extracted cells, isolated cell walls and glucan preparations made from them were effective inducers, but living yeast cells or cells killed by minimal heat treatment were not. 3. Chromatographic fractionation of lytic culture fluids showed the presence of two types of endo-β-(1→3)-glucanase and several β-(1→6)-glucanases; the latter may be induced separately by growing the myxo-bacterium in the presence of lutean. 4. Extensive solubilization of yeast cell walls was obtained only with preparations of one of these glucanases, an endo-β-(1→3)-glucanase producing as end products mainly oligosaccharides having five or more residues. Lysis by the other endo-β-(1→3)-glucanase was incomplete. 5. The β-(1→6)-glucanases produced a uniform thinning of the cell walls, and mannan–peptide was found in the solution. 6. These results, and the actions of the enzyme preparations on a variety of wall-derived preparations made from baker's yeast, are discussed in the light of present conceptions of yeast cell-wall structure.

Effects of Carbohydrate Pulses and pH on Population Shifts within Oral Microbial Communities in vitro

1989 ◽  
Vol 68 (9) ◽  
pp. 1298-1302 ◽  
Author(s):  
D.J. Bradshaw ◽  
A.S. McKee ◽  
P.D. Marsh
Keyword(s):  
Microbial Communities ◽  
Carbohydrate Metabolism ◽  
Oral Bacteria ◽  
The Other ◽  
Carbohydrate Availability ◽  
Oral Microflora ◽  
End Products ◽  
Per Se

A mixed culture chemostat system was used to distinguish between the effects of carbohydrate availability per se and the low pH generated from carbohydrate metabolism on the proportions of bacteria within microbial communities. Nine oral bacteria were grown at pH 7 and pulsed with glucose on ten consecutive days. In one chemostat, the pH was maintained automatically at 7 throughout the experimental period, while in the other, pH control was discontinued for six hours after each pulse. Glucose pulses at neutral pH had little effect on the composition of the microflora. Only the proportions of A. viscosus and V. dispar increased; L. casei and S. mutans remained at low levels (0.2% and 1.0%, respectively). Acetate and propionate were the predominant end-products of metabolism; lactate levels were low. In contrast, when pH was allowed to fall after each glucose pulse, the composition of the microflora altered dramatically. The amounts of L. casei and S. mutans increased both as a proportion of the total count and in absolute numbers, as did V. dispar, whereas the amounts of the other Gram-negative organisms (B. intermedius, F. nucleatum, and N. subflava) and S. sanguis were considerably reduced. Lactate formed a major portion of the metabolic end-products. Successive glucose pulses resulted in both amplified changes in the microflora and a steadily greater rate and final extent of acid production. This is in agreement with the reported shifts in the oral microflora in vivo in response to frequent carbohydrate intake. Analysis of the data strongly suggests that the pH generated from carbohydrate metabolism, rather than carbohydrate availability per se, is responsible for the widely reported shifts in composition and metabolism of the oral microflora in vivo.

THE BIOSYNTHESIS OF 11-KETOTESTOSTERONE AND 11β-HYDROXYTESTOSTERONE BY ATLANTIC SALMON TISSUES IN VITRO

1967 ◽  
Vol 45 (4) ◽  
pp. 581-589 ◽  
Author(s):  
D. R. Idler ◽  
H. C. Macnab
Keyword(s):  
Atlantic Salmon ◽  
Testicular Tissue ◽  
In Vitro Synthesis ◽  
Gonadal Tissue ◽  
First Time

The in vitro synthesis of 11-ketotestosterone and 11β-hydroxytestosterone, from adrenosterone and testosterone, by gonadal and interrenal slices and by sperm was studied. The results establish that adrenosterone is the major precursor of 11-ketotestosterone in both interrenal and testicular tissue. 11β-Hydroxytestosterone was synthesized mainly from testosterone by gonadal and interrenal tissue. The production of 11β-hydroxytestosterone by normal gonadal tissue is reported for the first time.

PATHWAYS FOR ANDROGEN SYNTHESIS IN VITRO BY THE TESTES OF JAPANESE QUAIL (COTURNIX COTURNIX JAPONICA)

1972 ◽  
Vol 55 (3) ◽  
pp. 499-506 ◽  
Author(s):  
TAKAO NAKAMURA ◽  
YUICHI TANABE
Keyword(s):  
Japanese Quail ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Testicular Tissue ◽  
Coturnix Japonica ◽  
Coturnix Coturnix Japonica ◽  
Coturnix Coturnix ◽  
Androgen Synthesis ◽  
Metabolic Products

SUMMARY The microsomal fraction of testicular tissue from Japanese quail (Coturnix coturnix japonica) was incubated with 4-14C-labelled pregnenolone, progesterone, 17α-hydroxyprogesterone, androstenedione, dehydroepiandrosterone and testosterone and [7-3H]17α-hydroxypregnenolone in a medium containing either NAD or NADPH. The metabolic products were identified by their mobilities on thin-layer chromatograms, derivative formation after oxidation and acetylation of the metabolites, and recrystallization to constant specific activity. Three pathways for testosterone formation by the testes are suggested by the results: the first via progesterone, 17α-hydroxyprogesterone and androstenedione; the second via 17α-hydroxypregnenolone, dehydroepiandrosterone and androstenedione; and the third via 17α-hydroxypregnenolone, dehydroepiandrosterone and androst-5-ene-3β,17β-diol.

scholarly journals Rescue and Conservation of Male Adult Alpacas (Vicugna pacos) Based on Spermatogonial Stem Cell Biotechnology Using Atomized Black Maca as a Supplement of Cryopreservation Medium

2021 ◽  
Vol 8 ◽  
Author(s):  
Martha Valdivia ◽  
Zezé Bravo ◽  
Jhakelin Reyes ◽  
Gustavo Gonzales
Keyword(s):  
Cell Viability ◽  
Antioxidant Properties ◽  
Testicular Tissue ◽  
The Other ◽  
Mitochondrial Activity ◽  
Isolated Cells ◽  
Male Adult ◽  
Testicular Cells ◽  
Other Hand

This is the first time that testicular tissue (n = 44) and isolated testicular cells (n = 51) were cryopreserved from alpaca testes 24 h postmortem. For this purpose, internally designed freezing media and cryopreservation protocols were used. Testicular tissue fragments (25 mg) and isolated testicular cells were frozen in MTDB (trehalose and black maca), MTD (trehalose), MSDB (sucrose and black maca), and MSD (sucrose) media. Isolated spermatogonial cells were cryopreserved in two ways, before and after proliferation in vitro. After cryopreservation, the percentage of cell viability in Group 1 (>50% of cell viability) by trypan blue did not show differences within each group (p > 0.05) but showed significant differences when comparing fragments with isolated cells (p < 0.05). Spermatogonial stem cells (SSC) were identified by flow cytometry as strong Dolichos biflorus agglutinin (sDBA) and mitochondrial activity of SSC as strongly positive for MitoSense (sMitoSense+) in intact mitochondria cells, weakly positive for MitoSense (wMitoSense+) in early apoptosis, and necrosis with 7-Aminoactinomycin-D positive (7-AAD). After freezing, in Group 1M (≥30% sMitoSense+), the fragments did not show differences between the media (p > 0.05), but in the isolated cells frozen in MSDB medium, 63.68 ± 8.90% (p < 0.05). In Group 2M (<30% sMitoSense+), necrosis (7AAD+) in MSDB medium was 27.03 ± 5.80%, and necrosis in isolated cells was 14.05 ± 9.3% with significant differences between these groups (p < 0.05); in sMitoSense+, the isolated cells (34.40 ± 23%) had a higher percentage than the fragments (12.4 ± 5.2) (p < 0.05). On the other hand, MSDB and MSD media were significantly higher for isolated cells than for fragments in sDBA+ (p < 0.05). On the other hand, the SSC (sDBA+) had significant differences (p < 0.05) between fresh cells 7.43 ± 1.3% (sDBA+) compared with those cryopreserved in MSDB medium 1.46 ± 0.34% (sDBA+). Additionally, the proliferated and cryopreserved SSC 6.29 ± 1.17% (sDBA+) did not show significant differences concerning the fresh cells (p > 0.05). In conclusion, the black maca showed antioxidant properties when it was included in the freezing medium and, therefore, improved the SSC's conservation of the alpaca. Furthermore, the proliferation of isolated cells in vitro produces a higher amount of SSC after thawing them for further preclinical or clinical work.

scholarly journals Matrix γ-Carboxyglutamic Acid Protein Is a Key Regulator of PTH-Mediated Inhibition of Mineralization in MC3T3-E1 Osteoblast-Like Cells

Endocrinology ◽  
2001 ◽  
Vol 142 (10) ◽  
pp. 4379-4388 ◽  
Author(s):  
Rajaram Gopalakrishnan ◽  
Hongjiao Ouyang ◽  
Martha J. Somerman ◽  
Laurie K. McCauley ◽  
Renny T. Franceschi
Keyword(s):  
Extracellular Matrix ◽  
Time Course ◽  
The Other ◽  
Activity Levels ◽  
Mrna Levels ◽  
Acid Protein ◽  
Carboxyglutamic Acid ◽  
Dependent Inhibition

Abstract As part of its overall function as a major regulator of calcium homeostasis, PTH stimulates bone resorption and inhibits osteoblast-mediated biomineralization. To determine the basis for the inhibitory actions of this hormone, we compared the time course of PTH-dependent inhibition of mineralization in MC3T3-E1 osteoblast-like cells with changes in mRNA levels for several extracellular matrix proteins previously associated either with induction or inhibition of mineralization. Mineralizing activity was rapidly lost in PTH-treated cells (∼30% inhibition after 3 h, 50% inhibition at 6 h). Of the proteins examined, changes in matrix γ-carboxyglutamic acid protein were best correlated with PTH-dependent inhibition of mineralization. Matrix γ-carboxyglutamic acid protein mRNA was rapidly induced 3 h after PTH treatment, with a 6- to 8-fold induction seen after 6 h. Local in vivo injection of PTH over the calvaria of mice also induced a 2-fold increase in matrix γ-carboxyglutamic acid protein mRNA. Warfarin, an inhibitor of matrix γ-carboxyglutamic acid protein γ-carboxylation, reversed the effects of PTH on mineralization in MC3T3-E1 cells, whereas vitamin K enhanced PTH activity, as would be expected if a γ-carboxyglutamic acid-containing protein were required for PTH activity. Levels of the other mRNAs examined were not well correlated with the observed changes in mineralization. Osteopontin, an in vitro inhibitor of mineralization, was induced approximately 4-fold 12 h after PTH addition. Bone sialoprotein mRNA, which encodes an extracellular matrix component most frequently associated with mineral induction, was inhibited by 50% after 12 h of PTH treatment. Osteocalcin mRNA, encoding the other known γ-carboxyglutamic acid protein in bone, was also inhibited by PTH, but, again, with a significantly slower time course than was seen for mineral inhibition. Taken together, these results show that the rapid inhibition of osteoblast mineralization induced by in vitro PTH treatment is at least in part explained by induction of matrix γ-carboxyglutamic acid protein.

Abstract 15735: Neonatal Cardiac Regeneration Depends on IGF1R-signaling

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Thomas Schuetz ◽  
Theresa Dolejsi ◽  
Alexander Bild ◽  
Axel Bauer ◽  
Josef M Penninger ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Crucial Role ◽  
Time Course ◽  
Cardiac Regeneration ◽  
Left Ventricular ◽  
Neonatal Mouse ◽  
The Other ◽  
Post Injury

Introduction: In contrast to other adult tissues myocardium cannot be sufficiently regenerated following significant myocardial infarction (MI). Efficient cardiac regeneration was demonstrated in neonatal mouse myocardial injury models. Similar observations were reported in other neonatal mammals including newborn human babies suffering from MI. The mechanisms of neonatal mammalian cardiac regeneration remain unclear. Here we show the crucial role of IGF1R which was unraveled in a time-course transcriptome analysis of neonatal mouse hearts. Methods: IGF1R was specifically knocked-down (KD) in cardiomyocytes of wildtype postnatal day one (P1) mice using adeno-associated virus (rAAV9) delivered shRNAmirs. KD of Renilla served as control (REN-CTRL). 5x10 13 viral genomes per kg bodyweight were injected intrathoracally. Three batches of rAAV9 with different shRNAmirs were used for the IGF1R-KD groups. Viral transduction was confirmed by bioluminescence imaging on luciferase containing rAAV9 copies and by immunofluorescence staining of rAAV9 delivered GFP reporter. KD efficiency was confirmed in vitro using a retrovirus system and in vivo. On P2 mice underwent either left anterior descending artery (LAD) ligation for induction of MI or SHAM surgery. Cardiac function was subsequently assessed by echocardiography 1 day post injury (dpi) and 21 dpi. Thereafter, hearts were harvested and left ventricular fibrosis was analyzed histologically. Results: LAD ligation resulted in significant MI in both, IGF1R-KD and REN-CTRL, LAD groups as proven by a markedly reduced ejection fraction (EF) 1 dpi. Importantly, 21 dpi IGF1R-KD and REN-CTRL SHAM groups displayed normal cardiac function proving no effect on neonatal cardiac growth and development of only KD of IGF1R without LAD ligation. In contrast, LAD ligation IGF1R-KD mice presented significantly reduced EF 21dpi compared to the other 3 groups. Histological analysis revealed significant fibrosis in the IGF1R-KD LAD hearts compared to the other 3 groups. Conclusions: Whereas IGF1R-KD or control rAAV9 does not alter physiological cardiac development, KD of IGF1R markedly impairs neonatal cardiac regeneration in neonatal mice after MI suggesting a crucial role in neonatal cardiac regeneration.

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