Growth differentiation factor‐9 improves development, mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles

2019 ◽  
Vol 54 (9) ◽  
pp. 1169-1176 ◽  
Author(s):  
Alane P. O. Monte ◽  
Jamile M. Santos ◽  
Vanúzia G. Menezes ◽  
Bruna B. Gouveia ◽  
Thae L. B. G. Lins ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Mitochondrial Activity ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9

Dynamic medium containing growth differentiation factor-9 and FSH maintains survival and promotes in vitro growth of caprine preantral follicles after long-term in vitro culture

2013 ◽  
Vol 25 (6) ◽  
pp. 955 ◽  
Author(s):  
A. M. C. V. Alves ◽  
R. N. Chaves ◽  
R. M. P. Rocha ◽  
L. F. Lima ◽  
P. M. Andrade ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Period ◽  
Oocyte Diameter ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Differentiation Factor ◽  
Follicle Diameter ◽  
Growth Differentiation Factor 9

The aim of the present study was to evaluate the effects of growth differentiation factor 9 (GDF-9) and FSH on the in vitro development of caprine preantral follicles cultured for 16 days. Ovarian fragments were cultured in αMEM+ (α-minimum essential medium, pH 7.2–7.4, 10 μg mL–1 insulin, 5.5 μg mL–1 transferrin, 5.0 ng mL–1 selenium, 2 mM glutamine, 2 mM hypoxanthine and 1.25 mg mL–1 bovine serum albumin) in the absence or presence of 200 ng mL–1 GDF-9 and/or 50 ng mL–1 FSH added during the first (Days 0–8) and/or second (Days 8–16) half of the culture period. Non-cultured and cultured fragments were processed for histological and ultrastructural analyses. After 16 days, all treatments using GDF-9 or FSH showed higher rates of follicular survival compared with αMEM+ alone. Compared with non-cultured control, sequential culture media containing GDF-9 and/or FSH significantly increased the percentage of developing follicles and follicle diameter. Moreover, a progressive increase in oocyte diameter was observed only with sequential culture medium containing GDF-9 until Day 8 followed by FSH (GDF-9/FSH) in the second half of the culture period. After 16 days of culture, ultrastructural analysis confirmed the integrity of follicles cultured in the presence of GDF-9/FSH. In conclusion, a dynamic medium containing GDF-9 and FSH (GDF-9/FSH) maintained follicular integrity and promoted activation of primordial follicles and growth during long-term in vitro culture of goat preantral follicles.

83 VITAMIN K2 SUPPLEMENTATION IMPROVES BLASTOCYST RATE BY RECOVERY OF MITOCHONDRIA IN IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 155
Author(s):  
L. Baldoceda ◽  
C. Vigneault ◽  
P. Blondin ◽  
C. Robert
Keyword(s):  
In Vitro Culture ◽  
Fluorescent Microscopy ◽  
Vitamin K2 ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Mitotracker Red

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.

206 EFFECT OF ADDITION OF FOLLICULAR FLUID OR GROWTH DIFFERENTIATION FACTOR-9 ON IN VITRO MATURATION OF PORCINE OOCYTES DENUDED 20h AFTER THE START OF IN VITRO MATURATION

2016 ◽  
Vol 28 (2) ◽  
pp. 234
Author(s):  
P. Ferré ◽  
T. T. M. Bui ◽  
M. T. Tran ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Dapi Staining ◽  
Experimental Conditions ◽  
Nuclear Maturation ◽  
Differentiation Factor ◽  
Growth Differentiation Factor 9

The interruption of communication between oocyte and cumulus cells (CC) can trigger meiotic resumption and exogenous additives, such as follicular fluid (FF) and growth differentiation factor-9 (GDF9), can improve oocyte quality and the developmental competence. This study was undertaken to examine if the absence and presence of FF from medium follicles (MF; 3–6 mm in diameter) or recombinant human GDF9 (Biovision, Milpitas, CA, USA) during the first or/and second half of in vitro maturation (IVM) had any effects on IVM of oocytes from small follicles (SF; 0.5–2 mm in diameter) or MF when the oocytes were denuded at 20 h after the start of IVM. Cumulus-oocyte complexes (COC) were aspirated from SF or MF of slaughtered prepubertal gilt ovaries. Groups of ~30 COC were cultured in a 300-μL drop of porcine oocyte medium containing 50 µM β-mercaptoethanol (mPOM) with or without 10% (v/v) FF and/or 100 ng mL–1 GDF9 at 39°C and 5% CO2 in air. During the first 20 h after the start of IVM, the medium was supplemented with 1 mM dibutyryl c-AMP, 10 IU mL–1 eCG and 10 IU mL–1 hCG. After the first period of IVM, the CC surrounding the oocytes were removed and the denuded oocytes continued culture for IVM with or without FF or/and GDF9 in the absence of dibutyryl c-AMP and gonadotropins in the same medium for another 24 h. At the end of IVM, meiotic progression of the oocytes was examined by DAPI staining. Statistical analyses from at least 4 replicates data were performed by a 2-way ANOVA and a Tukey’s multiple comparisons test. Removal of CC 20 h after the start of IVM significantly improved the incidence of mature oocytes derived from SF (59.2–64.1% v. 41.6–43.1% in controls, P < 0.05) but not from MF (73.1–78.5% v. 70.6–71.8% in controls), whereas regardless of supplementation with FF or GDF9, the maturation rates were always significantly higher in the denuded oocytes from MF (72.4–83.6%) than SF (57.8–66.2%; P < 0.05). Despite of the origin of COC (SF or MF), maturation rates of oocytes denuded 20 h after the start of IVM were not affected by supplementation with FF or GDF9 during the first and/or second half of IVM (P > 0.05). In summary, CC removal from COC 20 h after the start of IVM promotes nuclear maturation of oocytes from SF. Exogenous additives such as GDF9 and follicular fluid from MF do not seem to affect the promotion of nuclear maturation in our experimental conditions.

scholarly journals Expression of growth and differentiation factor 9 (GDF-9) and its effect on the in vitro culture of caprine preantral ovarian follicles

2011 ◽  
Vol 100 (2-3) ◽  
pp. 169-176 ◽  
Author(s):  
A.P. Almeida ◽  
M.V.A. Saraiva ◽  
V.R. Araújo ◽  
D.M. Magalhães ◽  
A.B.G. Duarte ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Ovarian Follicles ◽  
Differentiation Factor

Expression pattern of zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes in ovine oocytes and in vitro-produced preimplantation embryos

2008 ◽  
Vol 20 (8) ◽  
pp. 908 ◽  
Author(s):  
Daniela Bebbere ◽  
Luisa Bogliolo ◽  
Federica Ariu ◽  
Stefano Fois ◽  
Giovanni Giuseppe Leoni ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Bone Morphogenetic Protein ◽  
Preimplantation Embryos ◽  
Chain Reaction ◽  
Differentiation Factor ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Polymerase Chain ◽  
Bone Morphogenetic Protein 15

The expression patterns of four maternal effect genes (MEG), namely zygote arrest 1 (ZAR1), maternal antigen that embryo requires (MATER), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), were determined in ovine oocytes and in vitro-produced preimplantation embryos. The existence of ZAR1 and MATER in ovine species has not been reported previously. Reverse transcription–polymerase chain reaction was performed on germinal vesicle and IVM MII oocytes, as well as in in vitro fertilised and cultured two-, four-, eight- and 12/16-cell embryos, morulae and blastocysts. Quantification of gene expression by real-time polymerase chain reaction showed the highest abundance of all transcripts analysed in the immature oocyte. During the following stages of preimplantation development, the mRNAs examined exhibited different patterns of expression, but often significant decreases were observed during maturation and maternal–embryonic transition. The transcription of the four genes did not resume with activation of the genome.

75 Improvement of Developmental Competence of Bovine In Vitro-Produced Embryos by Using Charcoal:Dextran-Stripped Fetal Bovine Serum on In Vitro Culture Media

2018 ◽  
Vol 30 (1) ◽  
pp. 176
Author(s):  
A. Mesalam ◽  
R. Kong ◽  
B.-H. Choi ◽  
K.-L. Lee ◽  
B.-Y. Park ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Mrna Levels ◽  
Fetal Bovine

Serum has widely been used as a main supplement to embryo in vitro culture media as it contains embryotrophic factors. Charcoal:dextran treatment of fetal bovine serum (FBS) removes lipophilic chemicals and certain steroid hormones and growth factors. The objective of this study was to investigate the effects of charcoal:dextran-stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium (SOF-BE1 medium supplemented with 10% of serum) on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The experiment was conducted in 6 replicates (350 oocytes per group). The differences in embryo development, integrated optical intensity, and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P < 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% v. 36.85 ± 0.89%, respectively). The total number of cells per Day 8 blastocyst was not significantly different (P > 0.05) between the CDS FBS group (208.40 ± 14.77) and the HI FBS group (195.11 ± 19.15). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly increased mitochondrial activity, as identified by MitoTracker Green, and reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P < 0.05) higher after 24 h in the CDS FBS than in the HI FBS group (85.33 ± 4.84% v. 68.67 ± 1.20%). Quantitative reverse transcription PCR showed that the mRNA levels of lipid metabolism-related genes, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, and the cholesterol metabolism related gene hydroxymethylglutaryl-CoA reductase were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of survival gene sirtuin 1, antioxidant gene superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen–thawed blastocysts were significantly (P < 0.05) higher in the CDS FBS group than in the HI FBS group; however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. In conclusion, these data suggest that supplementation of in vitro culture medium with CDS FBS improves in vitro bovine embryo developmental competence and the quality of blastocysts in terms of their crytolerance and gene expression. This research was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets), BK21plus, and KGSP.

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.

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