Timing and regulatory aspects of oocyte maturation in vitro in the tammar wallaby (Macropus eugenii)

1999 ◽  
Vol 11 (5) ◽  
pp. 247 ◽  
Author(s):  
Karen E. Mate ◽  
Janine M. Buist
Keyword(s):  
Kinase Inhibitor ◽  
Chromatin Condensation ◽  
Calf Serum ◽  
Tammar Wallaby ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
Macropus Eugenii ◽  
Nuclear Maturation

Oocytes from a marsupial, the tammar wallaby (Macropus eugenii), resemble those of eutherian mammals in their ability to resume meiosis in vitro when cultured under suitable conditions. Culture for 42–48 h in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal calf serum, and 10 g mL –1 porcine luteinizing hormone (pLH) was required in order for oocytes, collected from the large antral follicles (> 2 mm diameter) of tammar wallabies (primed with 6 mg of porcine follicle stimulating hormone twice daily for four days), to proceed to metaphase II (MII) of meiosis. Under these conditions, chromatin condensation was observed within 4–8 h of culture in 61% of oocytes; metaphase I (MI) chromosomes were observed from 18–30 h of culture (66%); and most oocytes (76%) progressed to MII by 42 h in vitro. The addition of cycloheximide, a protein synthesis inhibitor, at concentrations of 1–100 g mL –1 , prevented maturation of tammar wallaby oocytes in vitro. This effect was reversible, as oocytes washed free of cycloheximide after 4 h of incubation were able to progress to MII. The addition of cycloheximide to wallaby oocytes at MI of meiosis prevented normal progression to MII suggesting that proteins critical for nuclear maturation are synthesized throughout the maturation process. Genistein, a protein kinase inhibitor decreased maturation of wallaby oocytes in a dose dependent manner. However, the concentration required to significantly inhibit maturation of wallaby oocytes (60 g mL –1 ) was greater than that required for eutherian species. Most wallaby oocytes were able to undergo germinal vesicle breakdown (GVBD) in the presence of high concentrations of genistein but produced abnormal chromatin configurations and were unable to progress to MII. Future studies will examine whether cytoplasmic changes occur in marsupial oocytes in vitro and their temporal relationship to nuclear maturation.

171 IN VIVO AND IN VITRO MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS–OOCYTE COMPLEXES DURING THE OVULATORY SEASON

2014 ◽  
Vol 26 (1) ◽  
pp. 199
Author(s):  
M. P. Cervantes ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
J. M. Palomino ◽  
G. P. Adams
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Bison Bison ◽  
Germinal Vesicle Breakdown ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Wood Bison

Technologies are being developed to conserve the genetic diversity of wood bison. Knowledge of the characteristics of in vivo and in vitro maturation of the cumulus–oocyte complex (COC) are needed in wood bison to design efficient in vitro embryo production protocols. The objectives were to (1) determine the optimal interval after hCG treatment for in vivo maturation of COC in superstimulated wood bison, and (2) compare the characteristics of COC after in vitro and in vivo maturation. Ovarian synchronization was induced in 25 bison during October and November by giving a luteolytic dose of prostaglandin followed 8 days later by follicular ablation (Day –1). Ovarian superstimulation was induced with FSH (Folltropin-V) given i.m. on Day 0 (300 mg) and Day 2 (100 mg). A second luteolytic dose of prostaglandin was given on Day 3. Bison were assigned randomly to 5 groups (n = 5/group). The COC were collected by transvaginal follicle aspiration on Day 4 and were either assessed immediately (0 h, control), or matured in vitro for 24 or 30 h (in vitro maturation), or collected on Day 5 (in vivo maturation), 24 or 30 h after bison were given 2000 IU of hCG i.m. on Day 4. In vitro maturation was done in TCM-199 with 5% calf serum, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin, at 38.5°C and in a 5% CO2 humidified atmosphere. Nuclear maturation was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII) with anti-lamin AC/DAPI staining. Groups were compared by analysis of variance and Fisher's exact test (Table 1). A mean (±s.e.m.) of 7.3 ± 1.7 COC were collected per bison, with no difference among groups. The COC in the control (0 h) group were at the nonexpanded GV stage. Cumulus cells were more expanded after in vivo than in vitro maturation, and the percentage of fully expanded COC was the highest in the 30-h in vivo maturation group (87%; P < 0.05). The greatest number of oocytes reached MII stage after 24 h of in vitro maturation, and 30 h of in vivo maturation. In conclusion, nuclear maturation occurred more quickly in vitro compared with in vivo, but the degree and incidence of cumulus expansion was greater after in vivo maturation. The competence of oocytes to undergo fertilization and develop into embryos remains to be investigated. Table 1.Cumulus expansion and nuclear maturation of wood bison oocytes

Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells

2002 ◽  
Vol 14 (7) ◽  
pp. 433 ◽  
Author(s):  
J. Ye ◽  
A. P. F. Flint ◽  
K. H. S. Campbell ◽  
M. R. Luck
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Synthesis Inhibitor ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Meiotic Progression ◽  
Conventional Culture ◽  
Oocyte Meiosis

This paper describes the use of the protein synthesis inhibitor cycloheximide (CHX) to synchronize nuclear progression during meiotic maturation in porcine oocytes, and also the time-dependence of nuclear maturation on exposure of the oocyte to cumulus cells. Prior to culture, the majority of oocytes were at the germinal vesicle (GV) stage (95–100%), but distributed from GVI to GVIV (GVI 56.1 ± 9.1%, GVII 15.3 ± 1.4%, GVIII 21.5 ± 7.1%, GVIV 7.1 ± 3.5%). During culture of cumulus-enclosed oocytes (COCs) from 12 h to 48 h in a conventional culture system, all meiotic stages were represented at any time point examined, with 63.6 ± 4.2% of oocytes maturing to metaphase II (MII). Cycloheximide blocked the progression of nuclear development in a dose-dependent manner. Treatment for 12 h with CHX at 1–25 μg mL–1 resulted in 95–100% oocytes being arrested and synchronized at GVII. With >5 μg mL–1 CHX, all oocytes were arrested before germinal vesicle breakdown (GVBD) (mostly at GVIII) by 24 h. A 12 h preincubation with 5 μg mL–1 CHX followed by 24 h of further culture without CHX resulted in >80% of oocytes maturing to MII. The profile of nuclear progression during maturation revealed discrete peaks of occurrence of different meiotic stages, with GVBD at 6–12 h, metaphase I (MI) at 10–18�h and anaphase I/telophase I at 16–20 h. After 12 h preincubation with 5 μg mL–1 CHX, denuded oocytes (DOs) matured to MI as COCs. However, DOs matured to MII as normal when denuded at MI. In conclusion, CHX not only efficiently blocks and synchronizes the meiotic progression of porcine oocytes at a specific GV stage, but it also effectively synchronizes subsequent meiotic progression to MII, resulting in discrete peaks of occurrence of different meiotic stages. Using this technique, the study showed that cumulus cells are essential for oocytes to mature from MI to MII but exposure to cumulus cells must occur before MI.

250 CYTOPLASMIC DYNEIN INTERMEDIATE CHAIN AND DYNACTIN p150Glued EXHIBIT DISTINCT SPATIAL AND TEMPORAL MICROTUBULE ASSOCIATIONS DURING BOVINE IN VITRO MATURATION AND ARE AFFECTED BY FOLLICLE SIZE

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
S. E. Racedo ◽  
M. C. Branzini ◽  
D. Salamone ◽  
V. Y. Rawe ◽  
H. Niemann
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Chromatin Condensation ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Meiotic Spindle ◽  
Germinal Vesicle Breakdown ◽  
Intermediate Chain

Microtubule molecular motors are critically involved in transporting vesicles during interphase, in building and maintaining spindles during mitosis and meiosis, and also in the localization of various organelles. DYNC1I1 (cytoplasmic dynein 1 intermediate chain) and its cofactor DCTN1 (dynactin p150Glued) are crucial for oocyte maturation but their role during mammalian female meiosis is not yet known. The goal of this study was to analyze the dynamics of these proteins in oocytes collected from different-size follicles at different stages of in vitro maturation (IVM), i.e., germinal vesicle stage (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII), and their association with microtubules. Ovaries were collected at a local abattoir. Cumulus–oocyte complexes (COCs) were aspirated from follicles either <2 mm or 2–8 mm in size and matured in M199, supplemented with 1% fatty acid-free BSA, 10 UI pregnant mare serum gonadotropin (PMSG)/5 UI HCG, and 100 µm cysteamine, at 39�C and 5% CO2. Follicle sizes and time points for fixation were: GV-0 h; GVBD-8 h for oocytes <2 mm and 9 h for oocytes 2–8 mm; MI-15 h; MII-24 h (Racedo et al. 2007, pub. online: 10.1002/mrd.20770). The distribution of the proteins was assessed by immunocytochemistry and laser confocal microscopy. The attached cumulus cells and zona pellucida of oocytes were removed in TALP-HEPES medium containing 1 mg mL–1 hyaluronidase and 2 mg mL–1 pronase, respectively. The oocytes were then incubated in a fixation–permeabilization solution containing 2% formaldehyde and 0.1%Triton X-100 for 1 h. Samples were then blocked for 1 h in 10 mm PBS + 0.3% BSA + 1% fetal calf serum (ICC blocking solution). The primary antibody was applied over night at 4�C, followed by treatment with fluorochrome-conjugated secondary antibodies for 1 h at 37�C in the dark. After RNase treatment, oocytes were incubated with TOTO-3 (Invitrogen, Carlsbad, CA, USA) to visualize the DNA. The material was mounted in an anti-fade medium (Vectashield�, Vector Laboratories, Burlingame, CA, USA) and imaged with a Zeiss laser scanning microscope. Immediately after chromatin condensation (GVBD), dynactin was in close association with the DNA and interacting with the spindles in MI and MII oocytes recovered from large follicles. No clear association with the DNA was observed in GVBD oocytes obtained from small follicles; little dynactin was found in MI and MII spindles. Dynein localization did not differ from dynactin in GVs and was homogeneously distributed in the cytoplasm of both groups of follicles. Dynein was not associated with the DNA in the GVBD stage while at MI and MII it was associated with the meiotic spindle. The association of dynein with microtubules was weak at the MI stage in oocytes from small follicles. Results provide insight into the regulatory mechanisms of oocyte maturation and a possible relationship with oocyte competence.

scholarly journals The effect of interaction between macromolecule supplement and oxygen tension on bovine oocytes and embryos cultured in vitro

Zygote ◽  
2009 ◽  
Vol 17 (4) ◽  
pp. 321-328 ◽  
Author(s):  
G.Z. Mingoti ◽  
V.S.D. Caiado Castro ◽  
S.C. Méo ◽  
L.S.S. Barretto ◽  
J.M. Garcia
Keyword(s):  
Oxygen Tension ◽  
Embryo Development ◽  
Calf Serum ◽  
Atmospheric Condition ◽  
Germinal Vesicle ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation

SummaryAiming to improve in vitro production of bovine embryos and to obtain supplements to replace serum for in vitro maturation (IVM), this study evaluated the effects of macromolecular supplementation of IMV medium (bovine serum albumin – BSA, polyvinyl alcohol – PVA, polyvinyl pyrrolidone – PVP, Ficoll, KnockoutSR, or fetal calf serum – FCS) and oxygen tension [5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2)] on oocyte maturation and embryo development. Nuclear progression to germinal vesicle breakdown, metaphase I and metaphase II stages were evaluated and overall results revealed that undefined (FCS) and semi-defined (BSA) media gave better results at 20% O2 and defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, IVM at 20% O2 was considered optimal for nuclear maturation. To evaluate embryo development, oocytes matured in the previously described conditions were fertilized and cultured at the same oxygen tension used for IVM and assessed for cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). Apart from oxygen tension, all treatments, except Knockout (22.7%), gave similar results for blastocyst development (26.5 to 38.7%). Independently of macromolecule supplement, higher development rates were obtained in an oxygen tension of 20% O2 (67.4% cleavage, 29.2% morulae, 40.8% blastocyst and 34.0% hatched blastocyst) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively). This study indicates that BSA, PVA, PVP and Ficoll can replace serum during IVM and that the optimal atmospheric condition for in vitro production of bovine embryos is 5% CO2 and 20% O2.

scholarly journals Role of leukemia inhibitor factor (LIF) in decidualisation of murine uterine stromal cells in vitro

2004 ◽  
Vol 181 (3) ◽  
pp. 477-492 ◽  
Author(s):  
AA Fouladi Nashta ◽  
CV Andreu ◽  
N Nijjar ◽  
JK Heath ◽  
SJ Kimber
Keyword(s):  
In Vitro Culture ◽  
Stromal Cells ◽  
Control Level ◽  
Calf Serum ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Alp Activity ◽  
Dose Dependent ◽  
In Cells

Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.

Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

2014 ◽  
Vol 26 (8) ◽  
pp. 1084 ◽  
Author(s):  
Yu-Ting Shen ◽  
Yue-Qiang Song ◽  
Xiao-Qin He ◽  
Fei Zhang ◽  
Xin Huang ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Maturation ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body ◽  
Triphenyltin Chloride ◽  
Dose Dependent

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 309-316 ◽  
Author(s):  
Carsten Krischek ◽  
Burkhard Meinecke
Keyword(s):  
Map Kinase ◽  
Protein Kinases ◽  
Chromatin Condensation ◽  
Specific Inhibitor ◽  
Histone H1 ◽  
Dependent Manner ◽  
Free Medium ◽  
Concentration Dependent Manner ◽  
Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

Biochemical studies of intrauterine components of the tammar wallaby Macropus eugenii during pregnancy

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 325-338
Author(s):  
Elizabeth J. Thornber ◽  
Marilyn B. Renfree ◽  
Gregory I. Wallace
Keyword(s):  
Protein Concentration ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Tammar Wallaby ◽  
Macropus Eugenii ◽  
Tenfold Increase ◽  
Specific Proteins ◽  
Wet Weight

The in vitro uptake and incorporation of [3H]ui idine by blastocysts of the tammar wallaby showed a 16- and 30-fold increase from day 0 to day 10 after removal of pouch young, respectively. Two of the six non-expanded blastocysts recovered on day 5 showed a tenfold increase in incorporation. During the first ten days after removal of pouch young the diameter of the blastocyst increased threefold. Endometrial exudate from gravid uteri had a higher protein concentration than exudate from nongravid uteri (39·5 ± 0·9 and 32·0 ± 2·0 mg/ml (mean ± s.e.m.), respectively). Endometrial exudates from uteri where the blastocyst was actively growing were found to contain six uterine-specific proteins. These were separated by gradient polyacrylamide gel electrophoresis. Two of the proteins were pre-albumins and the others were larger molecules (M.W. 153000–670000). Two proteins were only present at particular stages of pregnancy: the other four were present at all stages from diapause to birth, in exudate from gravid and nongravid uteri. The specific binding of progesterone and androstenedione to proteins in endometrial exudates or uterine flushings from pregnant wallabies was less than one per cent of the value obtained from day-5 pregnant rabbits. The ability of mouse blastocysts to take up and incorporate [3H]uridine into acidinsoluble material increased threefold in the presence of day-10 endometrial exudates from wallabies. However, this was less than ten percent of the values obtained in the presence of bovine serum albumin. The concentration of calcium in endometrial exudates increased from 23·6 to 45·2 μg/ml during pregnancy; in endometrium it remained at 88·7 μg/g (wet weight) throughout pregnancy, and in plasma it was 53·3 μg/ml. The concentration of zinc in endometrial exudates was 4·5 μg/ml; in endometrium it decreased from 21·8 to 13·3 μg/g (wet weight) during pregnancy and in plasma it was 0·6 μg/ml.

Maturation of porcine oocytes for 33 or 44 hours in vitro in the presence or absence of serum

1998 ◽  
Vol 1998 ◽  
pp. 180-180
Author(s):  
K. Rust ◽  
M.E. Staines ◽  
GJ. McCallum ◽  
N.S. Prathalingam ◽  
S.A. Edwards ◽  
...  
Keyword(s):  
Disease Transmission ◽  
Calf Serum ◽  
International Markets ◽  
Foetal Calf Serum ◽  
Maturation Time ◽  
Nuclear Maturation ◽  
Defined Media ◽  
Porcine Embryo ◽  
Disease Risks

Porcine embryo production in vitrois providing the impetus for the development of cryopreservation strategies aimed at welfare-friendly domestic and international marketing and movement of stock in a manner that minimises risks of disease transmission. In the context of disease risks, defined media, which avoid the use of serum and other biohazardous products, are likely to become essential in the production of embryos for international markets. In preparing for this situation, the present comparative study investigated in vitronuclear maturation of porcine oocytes in the presence of either foetal calf serum (FCS) or polyvinyl alcohol (PVA). In addition, the effect of restricting the maturation time to 33 rather than 44 hours was examined.

scholarly journals PPARγ regulates adipose triglyceride lipase in adipocytes in vitro and in vivo

2007 ◽  
Vol 293 (6) ◽  
pp. E1736-E1745 ◽  
Author(s):  
Erin E. Kershaw ◽  
Michael Schupp ◽  
Hong-Ping Guan ◽  
Noah P. Gardner ◽  
Mitchell A. Lazar ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Adipose Tissue ◽  
Lipid Metabolism ◽  
Protein Synthesis Inhibitor ◽  
Adipose Triglyceride Lipase ◽  
Dependent Manner ◽  
Triglyceride Lipase ◽  
Brown Adipose

Peroxisome proliferator-activated receptor-γ (PPARγ) regulates adipocyte genes involved in adipogenesis and lipid metabolism and is the molecular target for thiazolidinedione (TZD) antidiabetic agents. Adipose triglyceride lipase (ATGL) is a recently described triglyceride-specific lipase that is induced during adipogenesis and remains highly expressed in mature adipocytes. This study evaluates the ability of PPARγ to directly regulate ATGL expression in adipocytes in vitro and in vivo. In fully differentiated 3T3-L1 adipocytes, ATGL mRNA and protein are increased by TZD and non-TZD PPARγ agonists in a dose- and time-dependent manner. Rosiglitazone-mediated induction of ATGL mRNA is rapid and is not inhibited by the protein synthesis inhibitor cycloheximide, indicating that intervening protein synthesis is not required for this effect. Rosiglitazone-mediated induction of ATGL mRNA and protein is inhibited by the PPARγ-specific antagonist GW-9662 and is also significantly reduced following siRNA-mediated knockdown of PPARγ, supporting the direct transcriptional regulation of ATGL by PPARγ. In vivo, ATGL mRNA and protein are increased by rosiglitazone treatment in white and brown adipose tissue of mice with and without obesity due to high-fat diet or leptin deficiency. Thus, PPARγ positively regulates ATGL mRNA and protein expression in mature adipocytes in vitro and in adipose tissue in vivo, suggesting a role for ATGL in mediating PPARγ's effects on lipid metabolism.

Sign in / Sign up

Export Citation Format

Share Document