Oviductal microvesicles and their effect on in vitro maturation of canine oocytes

The effect of conditioned medium (CM) or microvesicles (MVs), secreted by multicellular spheroids of oviductal cells, and the involvement of some microRNAs (miRNAs) were investigated in canine oocyte maturation. To generate CM, spheroids were cultured for 3 days. MVs were obtained by ultracentrifugation of CM at 100,000 gand measured for size and concentration by NanoSight instrument. Cumulus-oocyte complexes (COCs) were matured at 38.5°C with 5% CO2and 5% of O2in synthetic oviductal fluid (SOF) in biphasic systems: for 24 h, with 5.0 μg/mL of LH and for other 48 h with 10% oestrous bitch serum. SOF was used as control (CTR) or supplemented with 10% CM or 25–50–75–100–150 × 106 MVs/mL labeled with PKH-26. Results show that multicellular aggregates secreted shedding vesicles. By fluorescence microscopy, the incorporation of labeled MVs was visible only at 72 h in oocyte cytoplasm. These MVs had a positive effect (P < 0.05) on maturation rate (MII) at the concentration of 75 and 100 × 106 MVs/mL compared to CM and CTR (20.34% and 21.82% vs 9.09% and 8.66% respectively). The concentration of 150 × 106 MVs/mL provided only 9.26% of MII. The expression of three specific miRNAs (miR-30b, miR-375 and miR-503) was studied. The lower rate of MII with the higher concentration of MVs is possibly due to the high level of miR-375. In conclusion, the oviductal MVs could be involved in cellular trafficking during oocyte maturation and their possible usein vitrocould facilitate the exploitment of canine reproductive biotechnologies.

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Effect of follicle-stimulating hormone on Bligon goat oocyte maturation and embryonic development post in vitro fertilization

Veterinary World ◽

10.14202/vetworld.2020.2443-2446 ◽

2020 ◽

Vol 13 (11) ◽

pp. 2443-2446

Author(s):

Diah Tri Widayati ◽

Mulyoto Pangestu

Keyword(s):

In Vitro Fertilization ◽

Embryonic Development ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Follicle Stimulating Hormone ◽

Fertilization In Vitro ◽

Vitro Fertilization ◽

Maturation Rate ◽

Stimulating Hormone

Background and Aim: Bligon goat is a crossbreed between Etawah and Kacang goat. This crossbreed goat is mostly reared by small farmers. In vitro maturation allows female goat (does) contributes toward reproduction despite the fact that the animal has been slaughtered. The aim of this study was to determine the in vitro maturation rate of Bligon goat oocytes supplemented with follicle-stimulating hormone (FSH), and their ability for further embryonic development after in vitro fertilization. Materials and Methods: Experiment was conducted at the Laboratory of Animal Physiology and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, using Bligon goat ovaries obtained from local slaughterhouse around Yogyakarta. One thousand five hundred cumulus-oocyte complexes were matured for 24 h in tissue culture medium 199 supplemented with 50 IU/L FSH or without FSH (control). First, matured oocytes were evaluated its morphology based on the expansion of cumulus cells and PB1 extrusion. Next, 600 oocytes were then stained with 1% aceto-orcein to examine maturation based on changes in the configuration of chromosomes and nuclear membrane breakdown. Oocytes were considered mature when they reached metaphase II. To prove the ability of mature oocytes to develop into embryos, 900 oocytes were processed for fertilization in vitro. The data were analyzed using analysis of variance. Results: The results indicated that FSH supplementation significantly increased oocyte maturation rate (65.21±7.26 vs. 43.25±6.23%) as indicated by extrusion of PB1 and homologous chromosome pairing and lined in the equator. The rate of degeneration was lower in the FSH-supplemented medium (3.21±0.25 vs. 10.17±3.15%). The blastocyst stage of oocyte developed embryos was reached by 12.43±2.15% and 22.28±4.86% of the control and treatment groups, respectively. Conclusion: FSH supplementation significantly improves oocyte maturation and yields mature oocytes for future embryo development in vitro.

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Use of Melatonin in the In Vitro Production of Bovine Embryos

Revista Brasileira de Saúde e Produção Animal ◽

10.1590/s1519-9940210322020 ◽

2020 ◽

Vol 21 ◽

Author(s):

Alan da Silva LIRA ◽

Ricardo de Macedo CHAVES ◽

Felipe de Jesus MORAES JUNIOR ◽

Sergio Henrique COSTA JUNIOR ◽

Brenda Karine Lima do AMARAL ◽

...

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Control Group ◽

Bovine Embryos ◽

In Vitro Production ◽

Maturation Medium ◽

Significant Difference ◽

Maturation Rate ◽

Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

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176 Effect of epigallocatechin-3-gallate on bovine oocyte in vitro maturation, fertilization, and development

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab176 ◽

2019 ◽

Vol 31 (1) ◽

pp. 212

Author(s):

Y. Honkawa ◽

Y. Gen ◽

S.-H. Hyon ◽

C. Kubota

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Polar Body ◽

Sperm Concentration ◽

P Value ◽

Bovine Oocyte ◽

Vitro Fertilization ◽

Significant Difference ◽

Maturation Rate

Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols, and a strong antioxidant compound. Huang et al. (2018 Asian-australas. J. Anim. Sci.) reported that adding 50μM EGCG can improve the bovine oocyte maturation rate. In this research, we investigated the effect of EGCG supplementation on different periods in bovine IVF. Cumulus-oocyte complex (COC) collected from ovaries of slaughtered cows were cultured in maturation medium (20 to 30 oocytes per 100-µL droplet), which consisted of TCM-199 with Earle’s salts and 25mM HEPES supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1µg mL−1 oestradiol, 0.02mg mL−1 FSH, and antibiotics at 38.5°C in a humidified atmosphere of 5% CO2 in air for 24h (in vitro maturation, IVM). After IVM, COC were fertilized in the fertilization medium (modified Brackett-Oliphant media supplemented with 10 µgmL−1 heparin, 10mM caffeine, and 3mg mL−1 BSA) for 6h using semen of one bull at final sperm concentration of 1×107 mL−1 (IVF). After IVF, COC were denuded and cultured in culture medium [CR1aa supplemented with 10% (vol/vol) FBS and antibiotics] at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90%N2 for 8 days (in vitro culture, IVC). The EGCG was supplemented at 10, 25, 50, and 100M in IVM medium; 25 and 50 µM in IVF medium; and 50 and 100 µM in IVC medium. After 24h in IVM medium, COC were denuded by pipetting, fixed in 3:1 ethanol:acetic acid for 24h and then checked for nuclear and polar body by using aceto-orcein stain. After 18h in IVF, the pronucleus in zygote was fixed in 3:1 ethanol:acetic acid for 24h and checked by aceto-orcein staining. Embryo development was evaluated by counting the total number of embryos that had reached compacted morula by 6 to 8 days after IVF. Significant differences were analysed by chi-squared test and residual analysis. A P-value<0.05 was considered statistically significant. When EGCG was added to IVM, there was no significant difference of oocyte maturation rate between all concentrations (0v. 10v. 25v. 50v. 100 μM: 73.9% v. 56.7% v. 76.7% v. 72.7% v. 63.5%). When EGCG was added to IVF, there was no significant difference of fertilized rate (0v. 25v. 50 μM: 59.4% v. 73.7% v. 64.9%). When EGCG was added to IVC, there was no significant difference in development rate (0v. 50v. 100 μM: 26.2% v. 15.7% v. 22.0%). In this research, EGCG addition did not affect bovine in vitro fertilization.

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155 Oocyte Maturation in Lyophilized In Vitro Maturation Medium as a Method to Increase the Medium’s Shelf-Life

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab155 ◽

2018 ◽

Vol 30 (1) ◽

pp. 217

Author(s):

M. Rubessa ◽

D. Weisgerber ◽

S. Bessler ◽

J. Bertels ◽

B. Harley ◽

...

Keyword(s):

Shelf Life ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Polar Body ◽

Cleavage Rate ◽

Embryo Production ◽

Freeze Dried ◽

Maturation Rate ◽

The Impact

The in vitro production of bovine embryos has dramatically increased in recent years, and with it the demand of stable media with a long shelf-life. In this experiment we evaluated the impact of the freeze-dried in vitro maturation (IVM) medium (Mdry) on in vitro oocyte maturation. We compared the standard IVM and the Mdry media. Medium M199 was used as base for the IVM medium. The percentage of metaphase II oocytes and embryo production were evaluated. Media solutions (10 mL) were aliquoted into 50-mL conical tubes and lyophilized to form a powder concentrate using a Genesis freeze-dryer (VirTis, Gardener, NY, USA). Lyophilization consisted of a constant cooling from 20°C to –10°C at a constant rate of 1°C/min with a 2-h hold at –10°C before sublimation at 0°C. The Mdry medium was held at –80°C for 4 months (only serum and hormones were added before the incubation). When the IVM medium was rehydrated, the pH were adjusted to 7.4. The percentage of mature oocytes was evaluated after 24 h of maturation. The oocytes were stained with Hoechst 33342, and only oocytes with metaphase and a polar body were evaluated as matured. Abattoir-derived Holstein oocytes (n = 540) were in vitro matured (25–30/well in 400 µL) and fertilized with sexed semen, according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). The oocytes were split for analysis (432 were used for IVP and 108 for maturation rate) over 6 replicates. Twenty hours after IVF, presumptive zygotes were cultured in SOF medium at 39°C with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed. All recorded parameters were subjected to a Student’s t-test. The parameters compared were maturation rate, cleavage rate, blastocyst rate and the percentage of embryos cleaved. The α level was set at 0.05. All data were expressed as quadratic means and mean standard deviations. The results showed no differences between the 2 groups (75.9% v. 74.1%) (t = 0.37; SD = 12.69; P = 0.36; df = 5) when we compared the nuclear maturation; however, when we evaluated embryo production, we found the Mdry treatment had a higher cleavage percentage (t = 2.39; SD = 14.81; P = 0.02; df = 5) and total embryos produced (t = 2.49; SD = 5.6; P = 0.02; df = 5) compared with the control (Table 1.). These results showed that lyophilization can be a valid method to increase the shelf life of IVP media. More replicates must be done in order to understand why the freeze-dried media produced more embryos. Table 1.Mean (SD in parentheses) percentage cleavage and blastocysts

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Gene Expression and In Vitro Maturation of Sheep Oocytes using Bee Pollen and Bee Honey as Medium Supplements

International Journal of Agriculture and Biology ◽

10.17957/ijab/15.1708 ◽

2021 ◽

Vol 25 (03) ◽

pp. 615-622

Author(s):

Aaishah M. Kaabi

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Maturation Medium ◽

Maturation Rate ◽

Group 3 ◽

Najdi Sheep ◽

Group 1

This study was conducted to investigate the effects of raw honey obtained from black seed or Sider and honeybee pollen as an additive in sheep oocyte maturation medium on the oocyte maturation rate, changes in oocyte glutathione (GSH) levels and expression of developmental candidate genes (GDF-9, MPF, C-MOS, IGF-1, BAX). Healthy immature oocytes of Najdi sheep were cultured in a medium supplemented with 5.0% Sider or Nigella sativa (black seed) honey + 1.0 μg/mL honeybee pollen, and after 24 h of incubation, the effects on the improvement of in vitro oocyte maturation were evaluated. Results demonstrated that the mean oocyte maturation rate was the best in group treated with 5% N. sativa (Group 3) compared with group treated with Sider or N. sativa honey (Group 1A and B, respectively). Mean GSH level was higher in Group 3 oocytes (11.09 ± 0.29 nmol) than in Group 2 oocytes (honey alone; 10.93 ± 0.57; P ≤ 0.05). Mean GSH levels were significantly decreased in Group 1. Expression analysis of candidate genes showed significant upregulation of GDF-9, cyclin B, C-MOS and IGF 1 genes in Group 3 and downregulation of BAX compared with control Group 1. In conclusion, addition of 1.0 μg/mL honeybee pollen along with one of two types of bee honey (Sider and N. sativa) at 5% concentration to the in vitro maturation medium of Najdi sheep oocytes has a beneficial effect in improving the maturation rate and gene expression and increasing the glutathione concentration in matured oocytes. © 2020 Friends Science Publishers

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Different Methods for Inducing Final Oocyte Maturation When Employing Progesterone-Primed Ovarian Stimulation Protocols

10.21203/rs.3.rs-1002007/v1 ◽

2021 ◽

Author(s):

Yen-Ju Sung ◽

Liang-Hsuan Chen ◽

Tzu-Hsuan Chin ◽

Shang-Yu Huang ◽

Hsing-Tse Yu ◽

...

Keyword(s):

Oocyte Maturation ◽

Gnrh Agonist ◽

Ovarian Stimulation ◽

Ovarian Response ◽

Poor Ovarian Response ◽

Follicular Maturation ◽

Ovarian Hyper Stimulation Syndrome ◽

Maturation Rate ◽

High Level

Abstract Background Evidently, when undergoing GnRH-antagonist protocols, dual trigger has proven to produce not just better quality and quantity of oocytes but also pregnancy outcome. However, not much comparative studies have been published when PPOS protocol is used for ovarian stimulation. Can the same positive outcomes be expected after the patients have been exposed to the high level of progesterone required for PPOS protocols? Methods In this retrospective cohort study, patients undergoing PPOS protocols were separated into three groups based on the method employed for triggering final follicular maturation, which included: (a) human chorionic gonadotropin (hCG); (b) Gonadotropin-releasing hormone-agonist (GnRH-agonist); or (c)dual trigger (GnRH-agonist + hCG). Either in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) was utilized for fertilization. Assessment comprised of their dynamic hormone profiles, embryonic analysis, and clinical outcomes. Results Of the 344 recruited patients, those fulfilling the Bologna criteria as poor ovarian responders and showing Estradiol (E2)<1000 pg/ml on the day of triggering had higher oocyte maturation rate (82% vs 58%, p<0.05) when triggered with dual trigger (GnRH-agonist + hCG) than hCG alone. For the patients with E2> 6500 pg/ml on the day of triggering, none of the three triggering methods demonstrated a significant advantage regarding the number of oocytes, percentage of matured oocytes, and rate of oocytes at fertilization or cleavage stages. Conclusions Implementing dual trigger for stimulating final follicular maturation in patients undergoing PPOS protocols is debatable. For poor ovarian response (POR) patients, dual trigger appeared to yield higher percentage of matured oocytes. In contrast, for hyper-responders, methods of triggering oocyte maturation did not affect the percentage of matured oocytes or the qualities of the embryos. For this group of patients, therefore, the agent used should be one that would reduce the risks of ovarian hyper-stimulation syndrome (OHSS).

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187 9-cis RETINOIC ACID IMPROVES MATURATION RATE AND ALTERS GENE EXPRESSION OF IN VITRO-MATURED OOCYTES IN EGYPTIAN BUFFALO

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab187 ◽

2017 ◽

Vol 29 (1) ◽

pp. 202

Author(s):

A. Gad ◽

S. Abu Hamed ◽

M. Khalifa ◽

A. El-Sayed ◽

S. A. Swiefy ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Retinoic Acid ◽

Vitamin A ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Polar Body ◽

Control Group ◽

Maturation Rate

Retinoic acid, a metabolite of vitamin A, regulates oocyte maturation through multiple mechanisms, including gene expression modulation or preventing oxidative stress. Effects of retinoic acid during oocyte maturation have been reported in several species; however, there have been no studies illustrating these effects in buffalo. Therefore, the objective of this study was to investigate the influence of 9-cis retinoic acid (9-cisRA), an active metabolite of vitamin A, on maturation rate and gene expression during in vitro maturation of buffalo oocytes. Cumulus-oocyte complexes (n = 360) were aspirated from surface follicles of Buffalo ovaries collected from local abattoirs and transported to the laboratory in physiological saline (0.9% NaCl) containing antibiotics (100 µg mL−1 of streptomycin sulfate and 100 IU mL−1 of penicillin) and maintained at 30°C. Grade A cumulus-oocyte complexes (evenly granulated cytoplasm and surrounded by multiple layers of cumulus cells) were randomly divided into 4 groups (90 oocytes/group) and allocated in TCM-199 medium supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 50 μg mL−1 of gentamycin, and 10 μg mL−1 of FSH and contained 0 (control), 5, 50, or 200 nM of 9-cisRA for maturation. After 24 h, maturation rate was calculated as a percentage based on polar body extrusion. In addition, gene expression patterns were analysed for antioxidant related genes (SOD1, CAT, GPX4, HOMX1, and PRDX1) and oocyte quality-related genes (GDF9 and BMP15) using quantitative real-time PCR with GAPDH as a housekeeping gene. Fold changes (FC) were calculated using ΔΔCt method (FC ≥2; P < 0.05). The results showed that maturation rate (based on the extrusion of polar body) was significantly higher in 5 nM 9-cisRA oocyte group (49.4 ± 2.1%) compared with the control group (35 ± 1.8%); in contrast, the 200 nM 9-cisRA oocyte group showed the lowest maturation rate (27.2 ± 2.7%). However, the 50 nM 9-cisRA oocyte group showed no significant differences (31.2 ± 3.8%) compared with control group .Oocytes treated with 5 and 50 nM 9-cisRA during in vitro maturation showed significant up-regulation of SOD1 (3.4 and 3.08 FC), CAT (2.7 and 1.8 FC), and HOMX1 (4.5 and 4 FC), and significant down-regulation of BMP15 (−3.7 and −3.6 FC), respectively, compared with the control group. Moreover, GPX4, PRDX1, and GDF9 genes were highly expressed in the 50 nM compared with the control group (13.2, 10.4, and 1.8 FC, respectively). In contrast, the 200 nM 9-cisRA group showed significant down-regulation of CAT (−60.3 FC), GDF9 (−2.5 FC), and BMP15 (−9.7 FC) compared with the control group. In conclusion, these results suggested that a low concentration of 9-cisRA (5 nM) in maturation media can improves maturation rate of buffalo oocytes and up-regulates the expression of oxidative stress response-related genes.

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O-231 In vitro maturation of human immature (GV) oocytes after controlled ovarian hormonal stimulation with recombinant AMH in the maturation medium

Human Reproduction ◽

10.1093/humrep/deab128.055 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

J Bedenk ◽

N Jančar ◽

E Vrtačnik-Bokal ◽

I Virant-Klun

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Polar Body ◽

Time Lapse ◽

Human Oocyte ◽

Hormonal Stimulation ◽

Maturation In Vitro ◽

Maturation Medium ◽

Maturation Rate

Abstract Study question Does the addition of recombinant AMH to the in vitro maturation (IVM) medium improve the maturation of GV oocytes after controlled ovarian hormonal stimulation? Summary answer Our results show that the addition of recombinant AMH to the in vitro maturation medium improves the maturation rate of GV oocytes. What is known already Anti-Müllerian hormone (AMH) is an important hormone involved in the process of sex differentiation during embryonic development. At the transition to the 21. century, more and more researchers have studied the role of AMH in ovarian function, especially its impact on folliculogenesis. AMH is becoming one of the main biomarkers of ovarian reserve and ovarian-specific disease, however, little is known about its effect on human oocyte maturation. Therefore, we matured immature GV (germinal vesicle) oocytes in IVM medium with recombinant AMH to assess its effect compared to the conventional IVM procedure with FSH and hCG. Study design, size, duration In this two-year prospective study, we compared the maturation rate of four groups of immature (GV) oocytes matured in maturation medium with added i) AMH (n = 15), ii) AMH+FSH+hCG (n = 44), iii) FSH+hCG (conventional; n = 22), and iv) hormone-free maturation medium (control; n = 15). Each oocyte was matured in vitro for a maximum of 28 hours and monitored by time-lapse microscopy to assess the time of GV breakdown (MI) and extrusion of the polar body (MII). Participants/materials, setting, methods Ninety-six GV oocytes of 46 patients (aged < 38 years, involved in the ICSI programme) after short antagonist protocol of controlled ovarian hormonal stimulation were included after written informed consent. IVM of oocytes was performed in the MediCult IVM System (LAG and IVM medium, Cooper Surgical, Denmark) with added hormones, and in a CO2 incubator equipped with the PrimoVision time-lapse microscope (Vitrolife, Sweden). Main results and the role of chance IVM medium with added recombinant AMH gave the best result with all (100 %) oocytes matured in vitro. In conventional IVM medium with FSH and hCG, the oocyte maturation rate was poorer, with 68 % of oocytes matured in vitro. An even lower oocyte maturation rate (34 %) was observed in IVM medium with AMH, FSH and HCG, which might be explained by the antagonistic action of these hormones. In a group of control oocytes, 25 % of oocytes matured in vitro. The mean time to GV breakdown (MI stage) was 3.7 hours and to polar body release (MII stage) 20,5 hours. The time to MI stage was quite comparable in all groups of oocytes (3.5, 3.8 and 3.7 hours). There was a tendency for the polar body to be released later if AMH was added to the maturation medium (21.5 and 20.2 vs. 19.9 hours) but differences were not statistically significant, as revealed by Student’s t-test. In the control group of oocytes, these times were prolonged (4.2 and 22.2 hours) due to slow spontaneous maturation. These preliminary results demonstrate that AMH could directly affect the oocyte maturation in vitro. Limitations, reasons for caution The limitation is the relatively small number of oocytes included; GV oocytes accounted for less than 10 % of all oocytes in the in vitro fertilisation (ICSI) programme. Moreover, the proportion of GV oocytes spontaneously matured to MI stage before the start of the experiment and were therefore not included. Wider implications of the findings Based on our data, we believe that AMH directly affects human oocyte maturation in vitro. Despite the common knowledge that AMH regulates the recruitment of growing ovarian follicles, it appears that the addition of AMH to the maturation medium can improve the human oocyte maturation in vitro. Trial registration number 0120-546/2018/6

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299 EFFECT OF ESTRADIOL-17β AND FOLLICLE STIMULATING HORMONE ON THE IN VITRO MATURATION OF PORCINE OOCYTES DERIVED FROM SMALL FOLLICLES

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab299 ◽

2015 ◽

Vol 27 (1) ◽

pp. 238

Author(s):

Y. Li ◽

C. Moros ◽

M. J. Izquierdo-Rico ◽

R. Romar ◽

H. Funahashi

Keyword(s):

In Vitro Maturation ◽

Egf Receptor ◽

Transcript Abundance ◽

Cumulus Cells ◽

Fsh Receptor ◽

Egf Receptors ◽

Relative Transcript Abundance ◽

Maturation Rate ◽

Positive Effect

In porcine cumulus-oocyte complexes (COC) from middle follicles (MF: 3–6 mm in diameter), FSH is known to induce the resumption of meiosis and accompanied by transactivate of the EGF receptor and activation of MAPK3/1 in the cumulus cells. The aim of the current study was to examine the effect of oestradiol-17β (E2: 0.1 μg mL–1) or FSH on in vitro maturation (IVM) of porcine oocytes derived from small follicles (SF: 1–2 mm in diameter). The COC were aspirated from MF of porcine ovaries obtained at slaughterhouse and cultured for IVM in mPOM (with 1 mM dibutyryl cAMP, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG for 20 h and then without those for 24 h in an atmosphere of 5% CO2 in air at 39°C) after washing 3 times. The COC from SF, which were aspirated at the same time with COC from MF, were precultured in the absence or presence of E2 or E2 plus FSH for 6 h before IVM culture. After the culture, oocytes were denuded from cumulus cells with 0.1% (vol/vol) hyaluronidase and the meiotic stage was observed. Relative transcript abundance of FSH and EGF receptors of CC was also examined by real-time RT–PCR just after preincubation for 6 h. Statistical analysis of data from 3 to 5 replicates was analysed by ANOVA and Tukey's multiple comparison tests. Maturation rate of oocytes from SF (40.6 ± 3.1%) was significantly lower than that of oocytes from MF controls (78.8 ± 2.8%, P < 0.01). Preincubation in the presence of E2 alone and E2 plus 0.005 IU of FSH significantly increases the maturation rate of oocytes from SF (56.8 ± 1.5 and 55.7 ± 3.1%, respectively, P < 0.01), although the rate was still lower than MF controls. However, in the presence of E2 plus a higher concentration of FSH (0.05 and 0.5 IU), oocyte maturation rate was similar (36.3 ± 2.4 and 33.7 ± 1.9%, respectively) to SF controls and lower than those of E2 alone and E2 plus 0.005 IU of FSH groups. Relative transcript abundance of FSH receptor of CC increased (P < 0.01) during preincubation in the presence of E2, but decreased in the presence of 0.05 IU of FSH. There were no significant differences in the transcript abundance of EGF receptors among treatments during preincubation (P = 0.09). In conclusion, preincubation of COC from SF in the presence of E2 alone and E2 plus 0.005 IU of FSH improves the maturation rate of the oocytes, whereas the presence of FSH more than 0.05 IU mL–1 concealed the positive effect. These effects may be yielded by change in the relative transcript abundance of FSH receptor of COC through the treatments.

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339 EFFECTS OF 17-β ESTRADIOL AND CYSTEAMINE ON IN VITRO MATURATION OF BEEF CATTLE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab339 ◽

2010 ◽

Vol 22 (1) ◽

pp. 326

Author(s):

L. Guo ◽

B. Tang ◽

X. Ma ◽

F. Gao ◽

J. B. Zhang ◽

...

Keyword(s):

Beef Cattle ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Bovine Embryo ◽

Lh Receptor ◽

Supplement Group ◽

Significant Difference ◽

Maturation Rate ◽

Harmful Effects

IVM is a critical step in in vitro bovine embryo production. Some materials supplemented in the IVM medium could improve the maturation rate. It was reported that 17-β estradiol (E2) stimulated the FSH-induced follicular growth and expression of the LH receptor in mural granulosa cells, and cysteamine could enhance glutathione synthesis, protect cells against harmful effects caused by oxidative injuries. In order to optimize IVM system of beef cattle oocytes, effects of different supplements (E2 and cysteamine), and dose of supplements on IVM of beef cattle oocytes were investi- gated in this study. The COCs were collected and cultured in the basic IVM media (TCM-199 + 10% FCS + 10 μg mL-1 FSH + 30 μg mL-1 LH) supplemented with hormonesof 0 μg mL-1 (control), 1 μg mL-1, 2 μg mL-1, and 4 μg mL-1 E2, respectively. Statistical analyzes were performed using SPSS (version 9.0) one-way ANOVA. Oocyte maturation rates (mean ± SEM) of four supplement groups were 60.52 ± 1.4%, 74.91 ± 0.25%, 77.25 ± 2.08%, and 62.20 ± 1.87%, respectively. No statistical differences were observed (P > 0.05) between 1 μg mL-1 and 2 μg mL-1 E2 groups; a similar trend was seen between 0 μg mL-1 and 4 μg mL-1 E2 groups. However, oocyte maturation rates (74.91-77.25%) of groups of 1 μg mL-1 and 2 μg mL-1 E2 were significantly higher (P < 0.05) than those (60.52-62.20%) of the other two groups. When basic IVM medium was sup- plemented with 0 μg mL-1 (control), 50 μg mL-1, and 100 μg mL-1 cysteamine, oocyte maturation rates were 75.25 ± 0.25%, 80.56 ± 0.57%, and 76.83 ± 1.82%, respectively. No significant difference (P > 0.05) was observed among them. However, there was approximately five percent higher maturation rate in the supplement group with 50 μg mL-1 cysteamine than those of groups supplemented with 0 μg mL-1 and 100 μg mL-1 cysteamine. Our results indicated: i) the appropriate supplement of E2 (1 μg mL-1 to 2 μg mL-1) could improve in vitro maturation of beef cattle oocytes. Also, the excessive supplement of E2 (>4 μg mL-1) could inhibit the maturation; ii) appropriate supplement of cysteamine (50 μg mL-1) was benefit to the IVM of beef cattle oocytes. This work was supported by the grant from national support plan, China, No. 2007BAD55B03; Corresponding author: Z.Y. Li.

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