Evaluation of cortical granules and viability of canine oocytes during long-term in vitro maturation

Global average infertility rate is about 6– 12%, and in Vietnam at around 7.7%. As a result, there is a high demand for treatment, especially for female infertility. In vitro maturation (IVM) was evaluated and proven to be the most popular and promising at the moment. In long-term cultivation, the follicle was observed to extend, therefore, the usage of a supporting frame is quite necessary to maintain follicle’s natural sphere structure as well as completing the mature process. Amniotic membrane is an avascular membrane, composed of collagen, fibronectin, nidogen, proteoglycan, containing a big number of growth – factors with antimicrobial, anti-inflammatory, low immunogenicity and viscoelasticity properties. Amniotic hydrogel owns structure formed with thin fibers to help preserve the main component as collagen, which can turn to gel form at 37 degree Celsius. With those properties, amniotic hydrogel showed high potential as a scaffold for the follicle. When amniotic hydrogel is used as a scaffold for cultivating of secondary follicle (100 – 130 µm), the size of oocyte and follicle increased after 12 days of culturing, along with the formation of antrum. The results demonstrated the possibility to use amniotic hydrogel as a scaffold for the development of the secondary follicle.

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Plasminogen activator activity in cortical granules of bovine oocytes during in vitro maturation

Theriogenology ◽

10.1016/s0093-691x(02)00666-0 ◽

2002 ◽

Vol 57 (7) ◽

pp. 1897-1905 ◽

Cited By ~ 14

Author(s):

Constantinos A. Rekkas ◽

Urban Besenfelder ◽

Vitezslav Havlicek ◽

Emmanuel Vainas ◽

Gottfried Brem

Keyword(s):

Plasminogen Activator ◽

In Vitro Maturation ◽

Cortical Granules ◽

Plasminogen Activator Activity ◽

Bovine Oocytes

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336 MEIOTIC DEVELOPMENT AND CORTICAL GRANULES DISTRIBUTION IN CANINE OOCYTES DURING IN VITRO MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab336 ◽

2010 ◽

Vol 22 (1) ◽

pp. 324 ◽

Cited By ~ 3

Author(s):

M. De los Reyes ◽

D. Luna ◽

J. Palomino

Keyword(s):

In Vitro Maturation ◽

Germinal Vesicle ◽

Fluorescein Isothiocyanate ◽

Cumulus Cells ◽

Homogeneous Distribution ◽

Cortical Granules ◽

Dapi Staining ◽

Nuclear Maturation ◽

Cytoplasmic Maturation

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM Hepes, 10% FCS, 0.25 mM pyruvate, 10 IU mL-1 of hCG, 300 IU mL-1 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma. Supported by Grant FONDECYT 1080618.

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155 EFFECT OF CO-CULTURE WITH CUMULUS-DERIVED SOMATIC CELLS DURING IN VITRO MATURATION ON PORCINE CUMULUS–OOCYTE COMPLEXES AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab155 ◽

2014 ◽

Vol 26 (1) ◽

pp. 191 ◽

Cited By ~ 1

Author(s):

J. D. Yoon ◽

L. Cai ◽

S. U. Hwang ◽

Y. Jeon ◽

E. Kim ◽

...

Keyword(s):

Embryonic Development ◽

Mrna Expression ◽

In Vitro Maturation ◽

Somatic Cells ◽

The Other ◽

Control Group ◽

Cortical Granules ◽

Developmental Potential ◽

Significant Difference

The purpose of this study was to investigate the effects of co-culture with cumulus-derived somatic cells (CSC) during porcine in vitro maturation (IVM) and subsequent embryonic development after IVF. The CSC were cultured in Dulbecco's modified Eagle medium for 48 h with various numbers of cumulus-derived somatic cells (0.0, 2.5, 5.0, and 10.0 × 104), and then cultured in TCM-199 for 4 h before the oocytes were added. Cumulus-oocytes complexes from 3- to 6-mm follicles were matured in 500 μL of TCM-199, with eCG and hCG, for 22 h, and then cultured in M199 without hormones for 22 h. Each experiment consisted of at least 4 replicates. Statistical analyses were carried out using SPSS 17.0 software (SPSS Inc., Chicago, IL). Percentage data were compared by one-way ANOVA, followed by Duncan's multiple range test. Data were presented as means ± s.e.m. Differences were considered to be significant if the P-value was 0.05. After IVM, no significant difference (P < 0.05) was observed in nuclear maturation rate among the 0.0, 2.5, 5.0, and 10.0 × 104 groups (88.0 ± 2.37, 81.5 ± 2.17, 87.0 ± 1.98 and 86.0 ± 1.93%, respectively). The 2.5 × 104 group showed a significant (P < 0.05) increase in intracellular glutathione (GSH) levels compared with that of the other groups. Intracellular reactive oxygen species (ROS) levels of mature oocyte in all groups showed no significant differences. The developmental competence of matured oocytes in all groups was evaluated after IVF. The 2.5 and 5.0 × 104 groups showed significantly (P < 0.05) high cleavage rates (60.0 ± 4.7 and 64.52 ± 5.9%, respectively) compared with the 0 and 10.0 × 104 groups (43.15 ± 5.0 and 53.8 ± 5.0%, respectively). The 2.5 × 104 group showed a significantly (P < 0.05) higher BL formation rate (35.7 ± 2.9) than control group (21.0 ± 3.8%, respectively), and higher total cell number (127.25 ± 7.7) compared with the 0 and 10 × 104 groups (89.3 ± 4.0 and 92.6 ± 3.7, respectively). In the analysis of gene expression, IVF-BL derived from the 2.5 and 5.0 × 104 groups showed higher (P < 0.05) mRNA expression of PCNA, which is an essential component of the DNA replication and repair machinery and POU5F1 has been used to evaluate developmental potential in embryos. The 10.0 × 104 group showed higher (P < 0.05) mRNA expression of caspase-3 and Bak as known pro-apoptotic factors, compared with the control group IVF-BL. The results of cortical granules distribution which leads digesting sperm receptor proteins ZP2 and ZP3 to block polyspermy, showed that the 2.5 × 104 group was increased significantly (P < 0.05) compared with the other co-culture groups (13.7 ± 6.1, 29.2 ± 9.5, 18.3 ± 0.8 and 19.52 ± 5.3, respectively). In conclusion, co-culture with 2.5 × 104 cumulus-derived somatic cells during IVM improved the developmental potential of porcine IVF embryos by increasing the intracellular GSH level and distribution of cortical granules during oocyte maturation. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

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172 Expression of proliferation and apoptosis markers in cumulus cells surrounding matured and aged oocytes exposed to luteotropic factors during the second phase of in vitro maturation

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab172 ◽

2019 ◽

Vol 31 (1) ◽

pp. 210

Author(s):

I. Lebedeva ◽

O. Mityashova ◽

A. Smekalova ◽

E. Montvila ◽

G. Singina ◽

...

Keyword(s):

In Vitro Maturation ◽

Caspase 3 ◽

Cumulus Cells ◽

Treated Group ◽

Second Phase ◽

Proliferation And Apoptosis ◽

Oocyte Aging ◽

Apoptosis Markers

The quality and developmental capacity of mammalian oocytes depends on cooperation with surrounding cumulus cells. The functional state and activity of cumulus cells changes with oocyte maturation, especially during the oocyte transition from metaphase I (MI) to metaphase II (MII) stage. In the present work, effects of 3 luteotropic factors, progesterone (P4), prolactin (PRL), and LH, during the second phase of in vitro maturation (IVM) on the subsequent expression of proliferation and apoptosis markers in bovine cumulus cells surrounding matured and aged oocytes were studied. A total of 1532 cumulus-oocyte complexes (COC) were cultured for 12h in TCM-199 containing 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH at 38.5°C and 5% CO2. Thereafter, COC were transferred to the following IVM systems: (1) TCM-199 containing 10% FCS (Control 1) and (2) a monolayer of granulosa cells (GC) precultured for 12h in TCM-199 containing 10% FCS (Control 2). In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (50ng mL−1) or ovine LH (5μg mL−1); then, the COC were matured for next 12h. Half of the COC matured for 12h in both systems were cultured for an additional 24h in fresh TCM-199 containing 10% FCS to test long-term hormonal effects during oocyte aging. After culture, the cumulus expression of the proliferation marker proliferating cell nuclear antigen (PCNA) and the pro-apoptotic markers caspase-3 and Bax was assessed by the immunocytochemical method. The data from 4 to 5 replicates using 84 to 106 COC per treatment were analysed by ANOVA. After IVM in System 1, the rate of PCNA-positive cumulus cells was higher (P<0.05) in the PRL-treated group (41.3±1.6%) than in the control (34.6±2.3%) or LH-treated group (29.9±2.9%), but did not differ from that in the P4-treated group (38.2±4.8%). In the presence of GC (System 2), the respective rates did not change but were more variable. Aging of COC matured in both systems led to a 1.4- to 1.9-fold reduction in the proportion of the cells containing the proliferation marker PCNA (P<0.05). Meanwhile, none of the hormones tested had any long-term effect on the proliferative activity of senescent cumulus cells. The rate of cumulus cells expressing caspase-3 in different groups varied from 48.5±4.9 to 53.8±5.8% and did not depend on the hormones, IVM system, or oocyte aging. The proportion of the Bax-positive cells was also unaffected by luteotropic factors but increased 1.4 to 1.6 times (P<0.01) following 24h of COC aging. Our findings indicate that PRL can exert a short-term stimulatory action on the proliferative activity of bovine cumulus cells in the course of the second phase of IVM. Meanwhile, the cumulus expression of pro-apoptotic markers caspase-3 and Bax is not responsive to P4, PRL, or LH during the second step of IVM. The study was supported by the Russian Science Foundation (project 16-16-10069).

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Distribution of cortical granules and meiotic maturation of canine oocytes in bi-phasic systems

Reproduction Fertility and Development ◽

10.1071/rd14022 ◽

2015 ◽

Vol 27 (7) ◽

pp. 1082 ◽

Cited By ~ 5

Author(s):

Maricy Apparicio ◽

Giuliano Q. Mostachio ◽

Tathiana F. Motheo ◽

Aracelle E. Alves ◽

Luciana Padilha ◽

...

Keyword(s):

In Vitro Maturation ◽

Germinal Vesicle ◽

Meiotic Maturation ◽

Cortical Granule ◽

Cortical Granules ◽

Germinal Vesicle Breakdown ◽

System A ◽

Cytoplasmic Maturation ◽

Positive Effect

The aim of this study was to evaluate the influence of different bi-phasic systems with gonadotrophins and steroids on in vitro maturation rates of oocytes obtained from bitches at different reproductive stages (follicular, luteal, anoestrous). In System A (control) oocytes were matured for 72 h in base medium (BM) with 10 IU mL–1 human chorionic gonadotrophin (hCG), 1 μg mL–1 progesterone (P4) and 1 μg mL–1 oestradiol (E2); in bi-phasic System B oocytes were matured for 48 h in BM with hCG and for 24 h in BM with P4; in bi-phasic System C oocytes were matured for 48 h in BM with hCG, P4 and E2, and for 24 h in BM with P4; in System D, oocytes were cultured in BM without hormonal supplementation. Data were analysed by ANOVA. There was a positive effect of the bi-phasic systems on germinal vesicle breakdown, metaphase I and metaphase II rates, irrespective of reproductive status (P < 0.05). Bi-phasic systems were also beneficial for cortical granule distribution (an indication of cytoplasmic maturation) and its relationship to nuclear status: 74.5% of the oocytes cultured in System B and 85.4% of those cultured in System C presented both nuclear and cytoplasmic maturation (P < 0.001). The stage of the oestrous cycle did not influence maturation rates.

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