270. Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells

2005 ◽  
Vol 17 (9) ◽  
pp. 111
Author(s):  
T. E. Hickey ◽  
D. L. Marrocco ◽  
F. Amato ◽  
L. J. Ritter ◽  
R. J. Norman ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Tritiated Thymidine ◽  
Paracrine Signalling ◽  
Secreted Factors ◽  
Differentiation Factor ◽  
Porcine Granulosa Cells ◽  
Growth Differentiation Factor 9 ◽  
Growth Promoting

Androgens, acting directly through the androgen receptor (AR), are thought to promote granulosa cell (GC) growth in vivo, but generally inhibit growth and promote GC differentiation in vitro. We hypothesised that the growth-promoting action of androgens on GC requires paracrine signalling from the oocyte. To test this hypothesis, we cultured mural GC from small antral (1–3mm) pre-pubertal pig follicles in the presence or absence of denuded oocytes (DO) from the same follicles to examine whether mitogenic responses, determined by uptake of tritiated thymidine, to combinations of FSH, insulin like growth factor 1 (IGF1) and dihydrotestosterone (DHT; 500 nM), were influenced by oocyte-secreted factors (OSFs). To further explore the identity of such factors, we performed the same experiments, substituting recombinant mouse growth differentiation factor 9 (GDF9), a known OSF, for the DO. Alone, DHT induced a small (<2-fold), but consistent increase in IGF1-stimulated DNA synthesis. OSFs stimulated DNA synthesis in all experimental combinations, most significantly in the presence of IGF1 (P < 0.0001), and DHT enhanced (P<0.05) the stimulatory effect of OSFs in all instances. Like OSFs, GDF9 substantially increased IGF1-stimulated DNA synthesis (P < 0.0001), and again, DHT enhanced (P > 0.01) this effect. In further experiments, two AR agonists, testosterone (10-1000nM) and DHT (5–500 nM), dose-dependently augmented the mitogenic effect of OSFs or GDF9 in the presence of IGF1. Only the highest doses of androgen had an independent stimulatory effect; lower doses required the presence of an OSF(s). Antiandrogen (hydroxyflutamide) treatment, used to block AR activity, antagonized the androgen X GDF9 interaction. In conclusion, androgens, via activation of the AR, stimulate porcine GC proliferation in vitro by potentiating the growth-promoting effects of oocytes or GDF9. These signalling pathway interactions are likely to be important regulators of folliculogenesis in vivo and may cause the excess follicle growth that is observed in androgen-treated female animals.

scholarly journals The effect of chemotherapy on the kinetics and proliferative capacity of normal and tumorous tissues in vivo

Blood ◽  
1975 ◽  
Vol 45 (1) ◽  
pp. 107-118 ◽  
Author(s):  
SH Rosenoff ◽  
JM Bull ◽  
RC Young
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Colony Formation ◽  
Antitumor Agents ◽  
Tritiated Thymidine ◽  
Chemotherapeutic Agents ◽  
Proliferative Capacity

Abstract The proliferative state of a given tissue is a major determinant of its sensitivity to both phase-specific and cycle-specific chemotherapeutic agents. To study the extent of injury induced by antitumor agents to normal and tumorous tissues, a technique for following DNA synthesis as reflected in the incorporation of tritiated thymidine (3H-TdR) into DNA was compared to the conventional radioautographic technique of the labeling index (LI) and to the functional kinetic technique of granulocyte colony formation in vitro. Alterations in DNA synthesis induced by a single dose of cyclophosphamide in normal and tumorous tissues in vivo paralleled in many respects the changes seen when the more time-consuming techniques of the LI or granulocyte colony formation were employed. However, the recovery of granulocyte colony formation after cyclophosphamide therapy laged behind the recovery of DNA synthesis in the bone marrow, obscuring a kinetic event of potential therapeutic significance. The determination of DNA synthesis simultaneously in normal and tumorous tissues in vivo was easy to perform and supplied therapeutically pertinent results comparatively quickly.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles

2013 ◽  
Vol 25 (8) ◽  
pp. 1194 ◽  
Author(s):  
G. L. Vasconcelos ◽  
M. V. A. Saraiva ◽  
J. J. N. Costa ◽  
M. J. Passos ◽  
A. W. B. Silva ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Nuclear Antigen ◽  
Mrna Levels ◽  
Related Factors ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Before And After ◽  
Vitro Development

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e. hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL–1 from Days 0 to 6 and 500 ng mL–1 from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P < 0.05). Alone, 200 ng mL–1 GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL–1 bovine serum albumin, 10 µg mL–1 insulin, 5.5 µg mL–1 transferrin, 5 ng mL–1 selenium, 2 mM glutamine, 2 mM hypoxanthine and 50 μg mL–1 ascorbic acid (α-MEM+). Comparisons of uncultured (0.2 mm) and α-MEM+ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P < 0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

scholarly journals Differences in the participation of TGFB superfamily signalling pathways mediating porcine and murine cumulus cell expansion

Reproduction ◽  
2011 ◽  
Vol 142 (5) ◽  
pp. 647-657 ◽  
Author(s):  
Robert B Gilchrist ◽  
Lesley J Ritter
Keyword(s):  
Cumulus Cell ◽  
Luciferase Reporter ◽  
Signalling Pathways ◽  
Reporter Assay ◽  
Paracrine Signalling ◽  
Secreted Factors ◽  
Morphogenetic Protein ◽  
Mapk Signalling ◽  
Growth Differentiation Factor 9

It is widely held that mammalian cumulus cell (CC) expansion requires oocyte-paracrine signalling, however in three of the four species studied to date, CC expansion occurs in the absence of the oocyte. This study was conducted to examine the paracrine and SMAD/MAPK intracellular signalling mechanism mediating porcine CC expansion, and to compare these to the mouse. Cumulus–oocyte complexes (COCs) and oocyte-free complexes (OOXs) from pigs and eCG-primed mice were treated in vitro with FSH and a broad range of TGFB superfamily antagonists. Expansion of porcine COCs and OOXs was unaffected by neutralisation of growth differentiation factor 9, TGFB, activin A, activin B and a broad spectrum bone morphogenetic protein antagonist. A SMAD-responsive luciferase reporter assay confirmed that porcine oocytes secreted factors that activate SMAD3 and SMAD1/5/8 in granulosa cells, but murine oocytes activated SMAD3 only. Treatment of COCs with a SMAD2/3 phosphorylation inhibitor (SB431542) partially inhibited porcine CC expansion and expression of TNFAIP6, but ablated murine CC expansion. SB431542 was equally effective at attenuating porcine CC expansion in the presence or absence of the oocyte. By contrast, a SMAD1/5/8 phosphorylation inhibitor (dorsomorphin) had no effect on porcine or murine CC function. Inhibition of ERK1/2 and p38 MAPK signalling pathways prevented porcine COC expansion and expression of most matrix genes examined. The activation of CC SMAD signalling by oocytes, and the requirement of SMAD2/3 signalling for expansion, is notably contrasted in pigs and mice. Nonetheless, porcine CC SMAD2/3 signalling is likely to be needed for optimal matrix formation, possibly by facilitating essential MAPK signals.

scholarly journals The Effect of Differing Demands for Blood Cell Production on DNA Synthesis by Hemopoietic Colony-Forming Cells of Mice

Blood ◽  
1965 ◽  
Vol 26 (3) ◽  
pp. 296-308 ◽  
Author(s):  
A. J. BECKER ◽  
E. A. MCCULLOCH ◽  
L. SIMINOVITCH ◽  
J. E. TILL
Keyword(s):  
Dna Synthesis ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Tritiated Thymidine ◽  
Large Fraction ◽  
Control Mechanisms ◽  
Normal Spleen ◽  
Hemopoietic System

Abstract A technic capable of estimating the fraction of hemopoietic colony-forming progenitor cells in DNA synthesis in vivo has been described. The technic is based on the ability of tritiated thymidine to inhibit the growth of those colony-forming cells which, by virtue of their presence in the S-phase during a 20-minute incubation in vitro, in the presence of 500 µc./ml. of H3TdR, have incorporated large amounts of the nucleoside. The method has been applied to transplanted colony-forming cells proliferating in spleens of heavily irradiated recipients as well as to cells from normal fetal liver, normal marrow, and normal spleen. In situations where the hemopoietic system is expanding (fetal liver and regenerating transplants), a large fraction, 40-65 per cent, of the stem cells are in DNA synthesis. In the steady-state situations (adult marrow and spleen), the fraction of cells in DNA synthesis is almost imperceptible using this technic. It is concluded that control mechanisms which govern the rate of hemopoiesis operate, at least in part, by altering the generative cycle of blood-forming progenitor cells.

scholarly journals Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis

Reproduction ◽  
2010 ◽  
Vol 139 (1) ◽  
pp. 71-83 ◽  
Author(s):  
P M Lokman ◽  
Y Kazeto ◽  
Y Ozaki ◽  
S Ijiri ◽  
R Tosaka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mrna Levels ◽  
Gh Treatment ◽  
Oocyte Growth ◽  
Anguilla Australis ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9 ◽  
Pituitary Homogenate

In order to study the regulation of the growth differentiation factor-9 (gdf9) gene in a primitive teleost with semelparous life history, we cloned a cDNA encoding shortfinned eel Gdf9, expressed a partial peptide inEscherichia coli, and raised an antiserum to evaluate changes in Gdf9 expression during its pituitary homogenate-induced reproductive cycle. The effects ofin vivoandin vitroexposure to the androgen 11-ketotestosterone (11-KT), known to affect previtellogenic (PV) oocyte growth, were also determined. Furthermore, we investigated whether Gdf9 expression was metabolically gated by treating PV fish with recombinant GHin vivo. Immunoreactive proteins ofca. 52 and 55 kDa were identified by western blot analysis. Gdf9 message and protein were most abundant in PV oocytes, and peaked slightly earlier for mRNA than for protein. Captivity resulted in reducedgdf9mRNA levels, which were restored following pituitary homogenate treatment. As oocytes progressed through induced oogenesis, Gdf9 expression decreased. Neither 11-KT nor GH treatment affectedgdf9mRNA levels in PV fish, although GH could partially restore handling- or captivity-induced decreases ingdf9mRNA levels. Semelparous eels thus show an expression pattern of Gdf9 during oogenesis that is similar to that seen in other vertebrates, that appears responsive to handling or captivity stress, and whose control remains to be elucidated.

Premeiosis and premeiotic DNA synthesis in the left ovary of the female chick embryo

Development ◽  
1967 ◽  
Vol 18 (3) ◽  
pp. 299-304
Author(s):  
M. Callebaut
Keyword(s):  
Chick Embryo ◽  
Dna Synthesis ◽  
Germ Cells ◽  
Tritiated Thymidine ◽  
Modified Technique ◽  
Distinctive Structure ◽  
Series Of Experiments ◽  
Prophase Of Meiosis

Premeiotic DNA synthesis in the germ cells of the female mouse embryo has been studied by Peters, Levy & Crone (1962) and Crone, Levy & Peters (1965). In this species, with a close synchronization in germinal development, the process of oogenesis which is the period of multiplication by mitotic divisions of the germ cells (oogonia) seems to be followed rapidly (within 24 h) by the first step of the prophase of meiosis (see Borum, 1961). In previous work (Callebaut & Dubois, 1965; Callebaut, 1967) we have investigated DNA synthesis in the ovarian germ cells of the chick embryo, both in vitro and in vivo, by autoradiography following the incorporation of tritiated thymidine. In a new series of experiments with a modified technique, it has been possible to demonstrate that the nuclei of the germ cells in premeiotic S phase in the female chick embryo have a distinctive structure.

scholarly journals Immunocytochemical Localization of Atrial Natriuretic Peptide, Vessel Dilator, Long-acting Natriuretic Peptide, and Kaliuretic Peptide in Human Pancreatic Adenocarcinomas

2005 ◽  
Vol 53 (8) ◽  
pp. 989-995 ◽  
Author(s):  
Sabiha R. Saba ◽  
Amanda H. Garces ◽  
Linda C. Clark ◽  
John Soto ◽  
William R. Gower ◽  
...  
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Dna Synthesis ◽  
Natriuretic Peptide ◽  
Peptide Hormones ◽  
Anticancer Effects ◽  
Growth Promoting ◽  
Long Acting ◽  
Atrial Natriuretic

We recently found that four peptide hormones synthesized by the same gene completely inhibit the growth of human pancreatic adenocarcinomas in athymic mice. The present immunocytochemical investigation was designed to determine where in the adenocarcinomas these peptide hormones localize. Atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide localized to the cytoplasm and nucleus of the human pancreatic adenocarcinomas, which is consistent with their ability to decrease DNA synthesis in the nucleus of this cancer. In this first investigation of where these peptide hormones with anticancer effects localize in any cancer, these peptide hormones also localized to the endothelium of capillaries and fibroblasts within these cancers. This is the first demonstration of growth-inhibiting peptide hormones localizing to the nucleus, where they inhibit DNA synthesis and may interact with growth-promoting hormones that localize there as the etiology of their ability to inhibit the growth of adenocarcinomas both in vitro and in vivo.

Effect of inhibin on rat testicular desoxyribonucleic acid (DNA) synthesis in vivo and in vitro

1981 ◽  
Vol 98 (2) ◽  
pp. 312-320 ◽  
Author(s):  
P. Franchimont ◽  
F. Croze ◽  
A. Demoulin ◽  
R. Bologne ◽  
J. Hustin
Keyword(s):  
Dna Synthesis ◽  
Thymidine Incorporation ◽  
Specific Activity ◽  
Tritiated Thymidine ◽  
Biological Properties ◽  
Rete Testis ◽  
Thymidine Uptake ◽  
Adult Rats

Abstract. When injected in vivo 3 h before sacrifice or when incubated in vitro with testicular fragments for 3 h, tritiated thymidine, a reliable index of DNA synthesis and of mitotic activity, was incorporated into the DNA of differentiated spermatogonia, as shown by autohistoradiography. The maximum DNA specific activity was obtained in pubertal rats aged 42 days, weight 150 g. Two preparations of inhibin extracted from ram rete testis fluid (RTF) of different molecular weight (> 10 000 for RTF1 and < 5000 for RTF3) but which possess the same biological properties were investigated for their effect on thymidine uptake in vivo and in vitro. In vivo both preparations specifically inhibited tritiated thymidine incorporation into testicular DNA of pubertal animals (42 days). No change in thymidine uptake into hepatic DNA was observed. Tritiated thymidine incorporation into testicular DNA was lower in normal adult rats and in hypophysectomized pubertal animals. RTF1 and RTF3 did not affect thymidine incorporation in either case. The reasons for this lack of effect are discussed. In vitro, both preparations induced a dose-dependent decrease in DNA synthesis in testis fragments from rats aged 42 and 49 days. The preparations lost their in vivo and in vitro inhibitory effects when denatured by heating and trypsin digestion. The inhibin preparations probably reduced testicular DNA synthesis and spermatogonial multiplication by reducing FSH secretion in vivo but also had a direct effect on the germ cells as shown by the in vitro experiments. These in vivo and in vitro actions of inhibin preparations are similar to those of the testicular chalones. The relationship which might exist between inhibin and the chalones is discussed.

scholarly journals The effect of chemotherapy on the kinetics and proliferative capacity of normal and tumorous tissues in vivo

Blood ◽  
1975 ◽  
Vol 45 (1) ◽  
pp. 107-118
Author(s):  
SH Rosenoff ◽  
JM Bull ◽  
RC Young
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Colony Formation ◽  
Antitumor Agents ◽  
Tritiated Thymidine ◽  
Chemotherapeutic Agents ◽  
Proliferative Capacity

The proliferative state of a given tissue is a major determinant of its sensitivity to both phase-specific and cycle-specific chemotherapeutic agents. To study the extent of injury induced by antitumor agents to normal and tumorous tissues, a technique for following DNA synthesis as reflected in the incorporation of tritiated thymidine (3H-TdR) into DNA was compared to the conventional radioautographic technique of the labeling index (LI) and to the functional kinetic technique of granulocyte colony formation in vitro. Alterations in DNA synthesis induced by a single dose of cyclophosphamide in normal and tumorous tissues in vivo paralleled in many respects the changes seen when the more time-consuming techniques of the LI or granulocyte colony formation were employed. However, the recovery of granulocyte colony formation after cyclophosphamide therapy laged behind the recovery of DNA synthesis in the bone marrow, obscuring a kinetic event of potential therapeutic significance. The determination of DNA synthesis simultaneously in normal and tumorous tissues in vivo was easy to perform and supplied therapeutically pertinent results comparatively quickly.

scholarly journals EVIDENCE FOR AN ESSENTIALLY CONSTANT DURATION OF DNA SYNTHESIS IN RENEWING EPITHELIA OF THE ADULT MOUSE

1963 ◽  
Vol 18 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Ivan L. Cameron ◽  
Richard C. Greulich
Keyword(s):  
Dna Synthesis ◽  
Mammalian Cells ◽  
Adult Mouse ◽  
Tritiated Thymidine ◽  
Constant Duration ◽  
The Times ◽  
Time Required ◽  
Relationship Of

Tritiated thymidine autoradiography has been applied to several renewing epithelial tissues of the adult mouse in order to determine (a) the average time required for DNA synthesis; and (b) the temporal relationship of the synthesis period to the progenitor cycles of these populations. The average duration of DNA synthesis has been computed from curves describing the rates of appearance and disappearance of labeled metaphase figures in epithelia of colon, ileum, duodenum, esophagus, and oral cavity, in both normal and colchicine-treated animals. In general, application of colchicine does not significantly influence the derived values for DNA synthesis duration. The DNA synthetic time is remarkably similar in the tissues examined, despite wide differences in the times required for completion of the progenitor cycle (and for tissue renewal). Synthesis of DNA in these epithelial cells of the mouse requires approximately 7 hours. Agreement between this value and those derived by other investigators for mammalian cells in vivo and in vitro indicates that DNA synthetic time may be a temporal constant, of considerable potential utility to studies of cell proliferation. The advantages and shortcomings of this experimental approach to problems of cell population kinetics in vivo are discussed.

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