Human charcoal-stripped serum supplementation enhances both the stearoyl-coenzyme a desaturase 1 activity of cumulus cells and the in vitro maturation of oocytes

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P >0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

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Growth factors improve mouse oocyte developmental potential via increased MAPK and MTOR signaling activities in cumulus cells during in vitro maturation

Fertility and Sterility ◽

10.1016/j.fertnstert.2018.07.882 ◽

2018 ◽

Vol 110 (4) ◽

pp. e313 ◽

Cited By ~ 1

Author(s):

J.C. Becker ◽

R. Pasquariello ◽

S.K. Rajput ◽

W.B. Schoolcraft ◽

R.L. Krisher ◽

...

Keyword(s):

Growth Factors ◽

In Vitro Maturation ◽

Cumulus Cells ◽

Mtor Signaling ◽

Mouse Oocyte ◽

Developmental Potential

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Influence of cumulus cells on in vitro maturation and fertilization of equine oocytes

Theriogenology ◽

10.1016/0093-691x(96)84736-4 ◽

1996 ◽

Vol 45 (1) ◽

pp. 263 ◽

Cited By ~ 3

Author(s):

M.V. Marcos ◽

A.R. Spell ◽

M.D. Butine ◽

M.J. Arns

Keyword(s):

In Vitro Maturation ◽

Cumulus Cells

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Use of pregnancy‐associated plasma protein‐A during oocyte in vitro maturation increases IGF‐1 and affects the transcriptional profile of cumulus cells and embryos from Nelore cows

Molecular Reproduction and Development ◽

10.1002/mrd.23259 ◽

2019 ◽

Vol 86 (11) ◽

pp. 1694-1704

Author(s):

Alan B. Giroto ◽

Patrícia K. Fontes ◽

Fernanda F. Franchi ◽

Priscila H. dos Santos ◽

Eduardo M. Razza ◽

...

Keyword(s):

Plasma Protein ◽

In Vitro Maturation ◽

Protein A ◽

Cumulus Cells ◽

Transcriptional Profile

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Lysophosphatidic acid increases in vitro maturation efficiency via uPA-uPAR signaling pathway in cumulus cells

Theriogenology ◽

10.1016/j.theriogenology.2018.02.020 ◽

2018 ◽

Vol 113 ◽

pp. 197-207 ◽

Cited By ~ 3

Author(s):

Seon-Ung Hwang ◽

Kyu-Jun Kim ◽

Eunhye Kim ◽

Junchul David Yoon ◽

Kyu Mi Park ◽

...

Keyword(s):

Signaling Pathway ◽

Lysophosphatidic Acid ◽

In Vitro Maturation ◽

Cumulus Cells

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326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab326 ◽

2006 ◽

Vol 18 (2) ◽

pp. 270

Author(s):

C. Hanna ◽

C. Long ◽

M. Westhusin ◽

D. Kraemer

Keyword(s):

In Vitro Maturation ◽

Calf Serum ◽

Cumulus Cell ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Culture Conditions ◽

Germinal Vesicle Breakdown ◽

Significant Difference ◽

Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

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334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab334 ◽

2006 ◽

Vol 18 (2) ◽

pp. 275

Author(s):

H. S. Lee ◽

Y. I. Seo ◽

X. J. Yin ◽

S. G. Cho ◽

I. H. Bae ◽

...

Keyword(s):

In Vitro Maturation ◽

Uv Light ◽

Cumulus Cells ◽

Oocyte Activation ◽

Cell Stage ◽

Oocyte Competence ◽

Treatment Groups ◽

Maturation Medium ◽

Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

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249 FOLLICLE-STIMULATING HORMONE AND DIBUTYRYL cAMP TREATMENTS DURING IN VITRO MATURATION IMPROVE THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab249 ◽

2008 ◽

Vol 20 (1) ◽

pp. 204

Author(s):

R. Oishi ◽

Y. Isaji ◽

H. Imai ◽

M. Yamada

Keyword(s):

In Vitro Maturation ◽

Basal Medium ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Developmental Competence ◽

Control Medium ◽

Dibutyryl Camp ◽

Mouse Oocytes ◽

Junctional Communication

The high level of cyclic adenosine monophosphate (cAMP), which is provided to the oocytes from cumulus cells via gap junctional complexes in cumulus-enclosed oocytes (CEOs), is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of CEOs. However, whether intraoocyte cAMP during the period of in vitro maturation (IVM) affects postfertilization developmental competence of mouse oocytes still remains unclear. The aim of this study was to examine the effects of FSH or dibutyryl cAMP (dbcAMP) treatment during IVM on in vitro development of mouse oocytes after in vitro fertilization (IVF). Whether a junctional association between cumulus cells and the oocyte would be essential for a cytoplasmic maturation-promoting effect was also examined. CEOs were isolated from and eCG-primed 3-week-old ICR mouse by rupturing preovulatory follicles with needles in M16 medium with 5% FCS and essential and nonessential amino acids (basal medium). IVM media used were basal medium without (control) or with 100 µm dbcAMP or 1 IU mL–1 FSH. Carbenoxolone (100 µm, CBX), an inhibitor of gap junction, was used to inhibit a junctional association between cumulus cells and the oocyte. Denuded oocytes (DOs) were prepared by repeatedly pipetting in basal medium with 0.2% hyaluronidase. CEOs and DOs were cultured in IVM media at 37�C under 5% CO2 in air for 16.5 h, and then transferred to TYH medium (a modified Krebs-Ringer bicarbonate medium) containing 0.4% BSA, followed by insemination with capacitated sperm. After 6 h of IVF, inseminated oocytes were cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was estimated at 24 h and 120 h after IVF, respectively. All experiments were done in 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's protected least-squares difference (PLSD) test. When CEOs were matured in IVM media, the rates of postfertilization development to the 2-cell and blastocyst stages of oocytes matured in the control medium were very low(29% and 13%, respectively), whereas those of oocytes matured with FSH or dbcAMP significantly increased (FSH: 61% and 52%, dbcAMP: 63 and 57%, respectively, v. control; P < 0.05). Next, when CEOs were matured in basal medium with 1 IU mL–1 FSH and 100 µm CBX, the developmental rate to the 2-cell stage (56%) was similar to that in medium with FSH alone (61%) but the rate to the blastocyst stage (40%) was little lower compared with that in medium with FSH alone (52%), although not significantly different (P > 0.05). Furthermore, when DOs were matured in IVM media, the developmental rates to the blastocyst stage after IVF of the oocytes matured with FSH or dbcAMP significantly increased (FSH: 25%, dbcAMP: 15%; P < 0.05) compared with those in control medium (7%). Taken together, it is suggested that increasing the concentration of intraoocyte cAMP during the IVM period is important to improve the developmental competence after IVF of mouse oocytes, and that the competence is acquired in part in a cumulus-oocyte junctional communication-independent manner.

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