Protein phosphorylation in bovine oocytes following fertilization and parthenogenetic activation in vitro

This study examined the event of protein phosphorylation in bovine oocytes in response to sperm penetration and parthenogenetic activation. In vitro matured oocytes were labelled with [32P]orthophosphate at 3 h intervals from 3 h to 18 h or from 0 h to 12 h following in vitro fertilisation and parthenogenetic activation, respectively. The level of protein dephosphorylation, at approximately 43 kDa, was similar in fertilised and parthenogenetically activated bovine oocytes. However, the level of protein phosphorylation at 40 kDa, 23 kDa and 18 kDa was different between these two samples. There were no such changes of protein phosphorylation and dephosphorylation in the control oocytes. Further, by two-dimensional gel electrophoresis there is a difference in the level of protein phosphorylation at 18 kDa between the fertilised and activated oocytes. These results suggest that this protein phosphorylation may be related to the formation of the male pronucleus in bovine oocytes.

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Comparison of activation ability between feline and bovine oocytes

Acta Veterinaria Hungarica ◽

10.1556/avet.2013.032 ◽

2013 ◽

Vol 61 (4) ◽

pp. 491-494 ◽

Cited By ~ 2

Author(s):

Fuminori Tanihara ◽

Yukine Kaedei ◽

Zhao Namula ◽

Vien Luu ◽

Yoko Sato ◽

...

Keyword(s):

Chemical Treatment ◽

Mammalian Species ◽

Parthenogenetic Activation ◽

Activation Rates ◽

Bovine Oocytes ◽

Higher Sensitivity

Research comparing the activation sensitivity of oocytes to chemical treatment among mammalian species remains limited. We compared the activation ability of oocytes from bovine and feline ovaries when treated with ethanol alone, with ethanol and cycloheximide, and without any chemical treatment; the oocytes were then cultured for 72 h. After in vitro maturation (IVM), 5% of feline oocytes were activated and 1% were cleaved, whereas there were no prematurely activated bovine oocytes. Activation rates with ethanol and ethanol/cycloheximide were significantly higher (P < 0.01) in bovine oocytes than in feline oocytes (74.2% vs. 34.1% and 86.3% vs. 52.5%, respectively). Thus, our findings indicate that feline oocytes can be prematurely activated by the end of IVM, and that bovine oocytes may have a higher sensitivity of parthenogenetic activation to chemical treatment than do feline oocytes.

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242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab242 ◽

2008 ◽

Vol 20 (1) ◽

pp. 200

Author(s):

T. H. C. De Bem ◽

R. Rochetti ◽

P. R. L. Pires ◽

F. F. Bressan ◽

P. R. Adona ◽

...

Keyword(s):

In Vitro Culture ◽

Embryo Development ◽

Polar Body ◽

Hatching Rate ◽

Blastocyst Development ◽

Embryo Production ◽

Parthenogenetic Activation ◽

Bovine Oocytes ◽

Hatching Rates

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.

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164 EFFECTS OF CUMULUS CELLS AND PITUITARY HORMONES ON AGE-ASSOCIATED CELLULAR CHANGES DURING THE PROLONGED CULTURE OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab164 ◽

2014 ◽

Vol 26 (1) ◽

pp. 196

Author(s):

I. Lebedeva ◽

G. Singina ◽

A. Lopukhov ◽

N. Zinovieva

Keyword(s):

Fetal Calf Serum ◽

Calf Serum ◽

Cumulus Cells ◽

Pituitary Hormones ◽

Meiotic Arrest ◽

Parthenogenetic Activation ◽

Cellular Changes ◽

Prolonged Culture ◽

Bovine Oocytes

In vivo and in vitro aging of mature mammalian oocytes heavily reduces their quality and developmental capacity. Therefore, the knowledge of physiological factors modulating the speed of oocyte aging is of great importance for successful reproduction. The goal of the present research was to study effects of cumulus cells (CC) and two related pituitary hormones, prolactin (PRL) and growth hormone (GH), on the dynamics of age-associated cellular changes during the prolonged culture of bovine oocytes in vitro. Bovine cumulus–oocyte complexes (COC) were cultured for 20 h in the following maturation medium: TCM 199 containing 10% fetal calf serum, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. After IVM, COC were transferred to the aging medium consisting of TCM-199 supplemented with 10% fetal calf serum and cultured for 0, 12, 24, or 36 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL (Research Center for Endocrinology, Moscow, Russia) or 10 ng mL–1 recombinant bovine GH (Monsanto, St. Louis, MO, USA). A portion of in vitro-matured oocytes were denuded of their CC and cultured in the control aging medium. At the end of culture, the state of the nuclear material in oocytes and embryos was evaluated by Tarkowski's cytogenetic method. The number of oocytes undergoing spontaneous parthenogenetic activation in the respective groups was determined by summarising the numbers of embryos cleaved and oocytes reaching anaphase-II to telophase-II stages or containing a pronucleus. Destructive changes in CC were assessed using the morphological signs of apoptosis. The data from 3 to 5 replicates were analysed by ANOVA. In the control group of COC, a rise in the rate of metaphase-II (M-II) oocytes with abnormal chromosome configurations occurred by 12 h of aging [31.8 ± 4.6% (12 h) v. 17.5 ± 2.6% (0 h); P < 0.05] and persisted up to 36 h (70.4 ± 2.0%; P < 0.001). At the same time, the frequency of oocyte parthenogenetic activation markedly increased only between 0 and 36 h of aging (from 0% to 20.7 ± 3.4%; P < 0.001). The addition of PRL or GH to the aging medium or removal of CCs resulted in a decline in the rate of M-II oocytes with degenerative changes of chromosomes throughout the culture period (at least P < 0.05). Furthermore, PRL and GH reduced the frequency of the oocyte activation at 36 h of the prolonged culture (up to 5.4 ± 2.5 and 1.7 ± 1.7%, respectively; P < 0.01), although CC did not influence meiotic arrest at M-II. Meanwhile, the rate of degenerated CC steadily increased as the culture time increased from 0 h (10.3 ± 1.1%) to 36 h (22.7 ± 2.2%; P < 0.001) and was unaffected by both hormones. The data suggest that, in bovine COC, CC accelerate abnormal changes in the chromosomal structure of aging M-II oocytes, whereas PRL and GH may decelerate these changes and support meiotic arrest during the prolonged culture of in vitro-matured oocytes. This research was partially supported by RFBR (project no. 13-04-01888).

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Parthenogenetic activation by electric stimulus of bovine oocytes matured in vitro

Theriogenology ◽

10.1016/0093-691x(89)90278-1 ◽

1989 ◽

Vol 32 (4) ◽

pp. 569-576 ◽

Cited By ~ 14

Author(s):

T. Kono ◽

S. Iwasaki ◽

T. Nakahara

Keyword(s):

Electric Stimulus ◽

Parthenogenetic Activation ◽

Bovine Oocytes

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Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro

Zygote ◽

10.1017/s0967199403002156 ◽

2003 ◽

Vol 11 (2) ◽

pp. 119-129 ◽

Cited By ~ 5

Author(s):

R.C. Chian ◽

J.T. Chung ◽

K. Niwa ◽

M.A. Sirard ◽

B.R. Downey ◽

...

Keyword(s):

Protein Phosphorylation ◽

Gel Electrophoresis ◽

Germinal Vesicle ◽

Two Dimensional Gel Electrophoresis ◽

Germinal Vesicle Breakdown ◽

One Dimensional ◽

Cell Cycle Transition ◽

Pronuclear Formation ◽

Bovine Oocytes

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

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