Follicular size affects the meiotic competence of in vitro matured prepubertal and adult oocytes in sheep

The current in vitro maturation system (IVM) for dog oocytes is inefficient. On the average, only 15% of ovarian oocytes complete nuclear maturation in vitro. For unknown reasons, the ability of oocytes to develop to the metaphase II stage (MII) varies markedly among bitches (Songsasen et al., 2002, Mol. Reprod. Dev. 62, 407–415). The objective of this study was to identify the cause(s) underlying these significant variations in nuclear maturation. Initially, we retrospectively analyzed data obtained during the past 3 years;; 1661 oocytes were obtained from 74 bitches where stage of reproduction for the donor was known based on ovarian morphology. Oocytes were cultured in TCM 199+0.1% polyvinyl alcohol at 38.5°C in 5% CO2 in humidified air under various experimental conditions. Analysis of variance (ANOVA) was performed to compare differences in meiotic competence of oocytes obtained at various reproductive stages and during different seasons. Stage of reproduction did not influence meiotic abilities of oocytes. Percentages of oocytes obtained during proestrus/estrus (n=468 oocytes), diestrus/metestrus (n=333), anestrus (n=331) or prepuberty (6–8 months of age, n=479) and developing to MII were 17.9±2.9%, (mean±SEM), 24.0±6.0%, 20.8±4.7%, and 17.8±5.2%, respectively (P>0.05). A similar analysis across seasons (spring, summer, fall, winter) also indicated no influence of time of year on nuclear maturation (P>0.05). Because there is a known strong link between follicular growth and meiotic competence of goat oocytes (De Smedt et al., 1994 J. Exp. Zool. 269, 128–139), we also examined the impact of follicular size on nuclear maturation. The cortex of ovaries from 15 bitches was horizontally dissected (5mm thickness) so follicles could be observed and divided into three classes: (1) <0.5mm diameter (n=60); (2) ≥0.5 to <1mm (n=110); and (3) 1–2mm (n=72). Follicles were separated according to these size classes;; oocytes were recovered and cultured in TCM 199+0.25mM pyruvate, 2mM glutamine, 25mM β-mercaptoethanol, 10ng/mL epidermal growth factor (Basal TCM) supplemented with 0.5IU/mL equine chorionic gonadotropin for 1h. Oocytes then were cultured in Basal TCM for 48h before staining with 1% orcein to assess nuclear status. Follicular size influenced meiotic competence of the oocytes (ANOVA, P<0.05). Mean percentages of MII oocytes were 14.2±7.2, 15.6±4.5, and 30.9±8.2, for oocytes recovered from <0.5-mm, ≥0.5 to <1-mm and 1–2-mm diameter follicles, respectively. This study revealed that stage of reproduction and season have no impact on in vitro nuclear maturation of the dog oocyte. However, the findings demonstrate that dog oocytes acquire meiotic competency during follicular development. Because the source of most dog oocytes for IVM are small follicles, results suggest that oocytes may be incapable of completing nuclear maturation under in vitro conditions that are designed for fully-grown oocytes.

Download Full-text

Cytochalasin B-induced pseudo-cleavage of mouse oocytes in vitro. II. Studies of the mechanism and morphological consequences of pseudocleavage

Journal of Cell Science ◽

10.1242/jcs.26.1.323 ◽

1977 ◽

Vol 26 (1) ◽

pp. 323-337

Author(s):

P.M. Wassarman ◽

T.E. Ukena ◽

W.J. Josefowicz ◽

G.E. Letourneau ◽

M.J. Karnovsky

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Cytochalasin B ◽

Oocyte Size ◽

Contractile Ring ◽

Subsequent Formation ◽

Mouse Oocytes ◽

Meiotic Competence ◽

Scanning Electron

Mouse oocytes are induced by cytochalasin B to undergo ‘pseudocleavage’ in vitro into 2 compartments, only one of which possesses microvilli. It has been found that this particular response to cytochalasin B is related to oocyte size and, possibly, to the acquisition of meiotic competence by the oocyte during its growth phase. Certain of the morphological events which characterize pseudocleavage have been determined using transmission and scanning electron microscopy. These events include: (i) an initial withdrawal of microvilli from the surface of the oocyte, together with the concomitant disappearance of microfilaments normally associated with the microvilli; (ii) the subsequent formation of a pseudocleavage furrow and contractile ring; and (iii) the reappearance of microvilli and associated microfilaments in one of the two resulting oocyte compartments. These changes in surface architecture are reflected in the distribution of fluorescein-conjugated lectins bound to the oocyte surface during pseudocleavage.

Download Full-text

Microfluidic Method of Pig Oocyte Quality Assessment in relation to Different Follicular Size Based on Lab-on-Chip Technology

BioMed Research International ◽

10.1155/2014/467063 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Bartosz Kempisty ◽

Rafał Walczak ◽

Paweł Antosik ◽

Patrycja Sniadek ◽

Marta Rybska ◽

...

Keyword(s):

Optical Fibers ◽

Spectral Characteristics ◽

Oocyte Quality ◽

Lab On Chip ◽

Chip Technology ◽

Porcine Oocyte ◽

Follicular Size ◽

On Chip ◽

Pig Oocyte

Since microfollicular environment and the size of the follicle are important markers influencing oocyte quality, the aim of this study is to present the spectral characterization of oocytes isolated from follicles of various sizes using lab-on-chip (LOC) technology and to demonstrate how follicle size may affect oocyte quality. Porcine oocytes (each,n=100) recovered from follicles of different sizes, for example, from large (>5 mm), medium (3–5 mm), and small (<3 mm), were analyzed after precedingin vitromaturation (IVM). The LOC analysis was performed using a silicon-glass sandwich with two glass optical fibers positioned “face-to-face.” Oocytes collected from follicles of different size classes revealed specific and distinguishable spectral characteristics. The absorbance spectra (microspectrometric specificity) for oocytes isolated from large, medium, and small follicles differ significantly (P<0.05) and the absorbance wavelengths were between 626 and 628 nm, between 618 and 620 nm, and less than 618 nm, respectively. The present study offers a parametric and objective method of porcine oocyte assessment. However, up to now this study has been used to evidence spectral markers associated with follicular size in pigs, only. Further investigations with functional-biological assays and comparing LOC analyses with fertilization and pregnancy success and the outcome of healthy offspring must be performed.

Download Full-text

51 Effect of Cryoprotectant Exposure Time on Development of Vitrified-Warmed Immature Equine Oocytes

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab51 ◽

2018 ◽

Vol 30 (1) ◽

pp. 164

Author(s):

H. S. Canesin ◽

J. G. Brom-de-Luna ◽

Y.-H. Choi ◽

A. M. Pereira ◽

G. G. Macedo ◽

...

Keyword(s):

Ethylene Glycol ◽

Propylene Glycol ◽

Exposure Time ◽

In Vitro Maturation ◽

Initial Exposure ◽

Exact Test ◽

Fetal Bovine ◽

Follicle Aspiration ◽

Meiotic Competence

Effective methods for cryopreservation of equine oocytes have not yet been established. Vitrification involves use of high cryoprotectant (CPA) concentrations, which can be cytotoxic. Thus, it is critical to determine a CPA concentration and exposure time able to protect the cell during cooling but with a minimal toxicity. Using a rapid non-equilibrating system, we fixed the time in the first, lower CPA concentration solution (V1) at 40 s, based on the time to maximal shrinkage. We then evaluated different exposure times in the final vitrification solution (V2). Cumulus-oocyte complexes (COC) were collected from slaughterhouse-derived ovaries and held overnight in commercial embryo holding medium. Fetal bovine serum was used as the base medium (BM). In experiment 1, COC were held in BM, incubated in V1 (2% propylene glycol + 2% ethylene glycol) for 40 s followed by incubation in V2 (17.5% propylene glycol + 17.5% ethylene glycol + 0.3 M trehalose) for 0, 45, 75, or 110 s, and then loaded in groups of 6 to 10 oocytes on a 75-µm steel mesh and plunged into liquid nitrogen. Warming was performed in decreasing trehalose concentrations in BM: 0.4 M (60-70 s), 0.2 M (5 min), 0.1 M (5 min), 0.05 M (5 min), and 0 M. After warming, oocytes were cultured for in vitro maturation (IVM) and evaluated after staining with Hoechst 33258. Differences between treatments were analysed by Fisher’s exact test. The maturation (metaphase II, MII) rate of the Control (non-vitrified oocytes; 38.8%, 31/80) was similar to that of the 75-s treatment (34.8%, 16/46; P = 0.71), and higher (P < 0.05) than those of the 0, 45, and 110 s treatments (0.0%, 0/10; 11.4%, 4/35; and 3.6%, 1/28; respectively). In experiment 2, timings in V2 focusing around 75 s were evaluated. The COC were collected and vitrified as for experiment 1, except that time in V2 was 50, 60, 70, 80, 90, or 100 s. The vitrified COC were then shipped to the intracytoplasmic sperm injection (ICSI) laboratory. After warming and IVM, oocytes were subjected to ICSI and embryo culture. Control oocytes were recovered by transvaginal follicle aspiration. The MII rate of the Control (60%, 33/55) was similar (P > 0.05) to that of the 60- and 70-s treatments (38.9%, 7/18, and 35.3%, 6/17, respectively), and higher (P < 0.05) than those of the 50-, 80-, 90-, and 100-s treatments (5.6 to 31.6%). The cleavage rates were 94% (31/33) for the Control and 71 to 100% for vitrified oocytes (P > 0.05). No blastocyst was produced from vitrified oocytes compared with 8/33 (24.2%) for Control. This work demonstrates that a rapid, non-equilibrating vitrification technique using a 40-s initial exposure and 70- to 80-s final exposure to CPA is associated with maintenance of meiotic competence of immature equine oocytes; however, further work is required to optimize embryonic development with this method. Research supported by the Clinical Equine ICSI Program and the Link Equine Research Fund, Texas A&M University.

Download Full-text

Expanded equine cumulus–oocyte complexes exhibit higher meiotic competence and lower glucose consumption than compact cumulus–oocyte complexes

Reproduction Fertility and Development ◽

10.1071/rd16441 ◽

2018 ◽

Vol 30 (2) ◽

pp. 297 ◽

Cited By ~ 7

Author(s):

L. González-Fernández ◽

M. J. Sánchez-Calabuig ◽

M. G. Alves ◽

P. F. Oliveira ◽

S. Macedo ◽

...

Keyword(s):

Glucose Metabolism ◽

Granulosa Cells ◽

Glucose Consumption ◽

Cell Types ◽

Solute Carrier Family ◽

Necrosis Factor Alpha ◽

Maturation Rate ◽

Solute Carrier ◽

Meiotic Competence

Equine cumulus–oocyte complexes (COCs) are classified as compact (cCOC) or expanded (eCOC) and vary in their meiotic competence. This difference could be related to divergent glucose metabolism. To test this hypothesis in the present study, eCOCs, cCOCs and expanded or compact mural granulosa cells (EC and CC respectively) were matured in vitro for 30 h, at which time maturation rate, glucose metabolism and the expression of genes involved in glucose transport, glycolysis, apoptosis and meiotic competence were determined. There were significant differences between eCOCs and cCOCs in maturation rate (50% vs 21.7% (n = 192 and 46) respectively; P < 0.001), as well as mean (± s.e.m.) glucose consumption (1.8 ± 0.5 vs 27.9 ± 5.9 nmol per COC respectively) and pyruvate (0.09 ± 0.01 vs 2.4 ± 0.8 nmol per COC respectively) and lactate (4.7 ± 1.3 vs 64.1 ± 20.6 nmol per COC respectively; P < 0.05 for all) production. Glucose consumption in EC and CC did not differ significantly. Expression of hyaluronan-binding protein (tumour necrosis factor alpha induced protein 6; TNFAIP6) was increased in eCOCs and EC, and solute carrier family 2 member 1 (SLC2A1) expression was increased in eCOCs, but there were no differences in the expression of glycolysis-related enzymes and solute carrier family 2 member 3 (SLC2A3) between the COC or mural granulosa cell types. The findings of the present study demonstrate that metabolic and genomic differences exist between eCOCs and cCOCs and mural granulosa cells in the horse.

Download Full-text

Nuclear morphology, diameter and meiotic competence of buffalo oocytes relative to follicle size

Reproduction Fertility and Development ◽

10.1071/rd03006 ◽

2003 ◽

Vol 15 (4) ◽

pp. 223 ◽

Cited By ~ 15

Author(s):

Muhammad Rizwan Yousaf ◽

Kazim Raza Chohan

Keyword(s):

Germinal Vesicle ◽

Cumulus Cells ◽

Nuclear Morphology ◽

Vitro Fertilization ◽

Size Dependent ◽

Size Groups ◽

Follicle Size ◽

Fetal Bovine ◽

Meiotic Competence

The nuclear morphology, diameter and in vitro meiotic competence of buffalo oocytes was compared relative to follicle size. Cumulus–oocyte complexes (COCs) were collected from 1–<2, 2–<3, 3–<4, 4–<6 and 6–<8 mm follicles from abattoir ovaries. Cumulus cells were removed using 3 mg mL−1 hyaluronidase in saline and repeated pipetting. Denuded oocytes were measured, fixed in 3% glutaraldehyde, stained with 4,6-diamidoino-2-phenylindole and evaluated for nuclear morphology, namely the stage of germinal vesicle (GV) development before in vitro maturation (IVM). The COCs from >2-mm follicles were matured in vitro in their respective size groups for 24 h in Medium 199 supplemented with 10 μg mL−1 follicle-stimulating hormone, 10 μg mL−1 luteinizing hormone, 1.5 μg mL−1 oestradiol, 75 μg mL−1 streptomycin, 100 IU mL−1 penicillin, 10 mM HEPES and 10% fetal bovine serum. Matured oocytes were fixed, stained and evaluated for GV status and meiotic development. The number of oocytes collected from follicles 1–<8 mm in diameter averaged 1.82 per ovary. Oocytes from follicles 1–<2 mm (107.7 ± 1.6 μm), 2–<3 mm (108 ± 1.1 μm) and 3–<4 mm (114.6 ± 1.3 μm) in diameter were smaller in diameter (P < 0.05) than oocytes from follicles 4–<6 mm (124.4 ± 1.3 μm) and 6–<8 mm (131.9 ± 1.4 μm) in diameter. A majority of oocytes (P < 0.05) from <4-mm follicles was at the initial stages of GV development (GV-I, II and III), whereas oocytes from 4–<6- and 6–<8-mm follicles were at the final stages of GV-IV (35.0 and 21.6% respectively) and GV-V (49.1 and 67.5% respectively). Poor IVM rates of 32.0% and 32.7% to metaphase (M)-II were observed for oocytes isolated from 2–<3- and 3–<4-mm follicles, respectively, whereas significantly (P < 0.05) more oocytes from 4–<6- and 6–<8-mm follicles reached M-II (67.1% and 79.1% respectively). In conclusion, buffalo oocytes displayed a size-dependent ability to undergo meiotic maturation and we suggest that oocytes from >4-mm follicles should be considered in buffalo in vitro fertilization systems for better results.

Download Full-text

The position of the germinal vesicle and the chromatin organization together provide a marker of the developmental competence of mouse antral oocytes

Reproduction ◽

10.1530/rep-09-0230 ◽

2009 ◽

Vol 138 (4) ◽

pp. 639-643 ◽

Cited By ~ 26

Author(s):

Michele Bellone ◽

Maurizio Zuccotti ◽

Carlo Alberto Redi ◽

Silvia Garagna

Keyword(s):

Germinal Vesicle ◽

Chromatin Organization ◽

Developmental Competence ◽

Cell Stage ◽

Morphological Marker ◽

Good Marker ◽

Chromatin Configuration ◽

Meiotic Competence

Based on their chromatin organization, antral oocytes can be classified into two classes, namely surrounded nucleolus (SN, chromatin forms a ring around the nucleolus), and not surrounded nucleolus (NSN, chromatin has a diffuse pattern). Oocytes of both classes are capable of meiotic resumption, but while SN oocytes, following fertilization, develop to term, NSN oocytes never develop beyond the two-cell stage. A recent study has shown that the position of the germinal vesicle (GV) can be used as a morphological marker predictive of oocyte meiotic competence, i.e. oocytes with a central GV have a higher meiotic competence than oocytes with an eccentric GV. In the present study, we have associated both markers with the aim of identifying, with more accuracy, the oocytes' developmental competence. Following their isolation, antral oocytes were classified on the basis of both SN and NSN chromatin configuration and their GV position, matured to metaphase II and fertilized in vitro. We demonstrated that the position of the GV is a good marker to predict the oocytes' developmental competence, but only when associated with the observation of the chromatin organization.

Download Full-text

Effect of culture methods on cumulus and oocyte morphology and meiotic competence of bovine oocytes from early antral follicles

Archives Animal Breeding ◽

10.5194/aab-48-562-2005 ◽

2005 ◽

Vol 48 (6) ◽

pp. 562-571

Author(s):

L. Kątska-Książkiewicz ◽

H. Alm

Keyword(s):

Three Dimensional ◽

Collagen Gel ◽

Standard System ◽

Dimensional Structure ◽

Culture Methods ◽

Normal Morphology ◽

Antral Follicles ◽

System A ◽

Meiotic Competence

Abstract. The objective of the present study was to establish a culture system that would maintain the three-dimensional structure of bovine early antral follicles (EAF) or isolated cumulus-oocyte-granulosa complexes (COCGs) and increase the resulting portion of COCs with normal morphology for subsequent IVM. The morphological quality and meiotic competence of oocytes originating from early antral bovine ovarian follicles (0.2 to 0.7 mm and 0.4 to 0.7 mm diameter) were evaluated following culture in vitro for 14 and 7 d, respectively, and subsequent in vitro maturation. Growth culture modifications included culture in the well of the well (WOW) system; a microdroplet of collagen gel (2 x 5 μl vs. 2 x 400 μl; standard system) and culture of EAF in hanging drops of medium (inverted system). Significantly higher (P<0.01) proportions of COCs with normal morphology (60.4%) were obtained from COCGs compared to EAF (4.8%) grown in the WOW system. Embedding of COCGs in microdrops of collagen gel significantly increased proportion of COCs with normal morphology (63.2%) compared to those embedded in standard volume gels (35.3%). Recovery rate of COCs with normal morphology from cultured EAF was improved both by using microdrops of gel (44%) and by culture in the inverted system (39.3%) over that found for the standard system (8.5%).

Download Full-text

Tingkat Kematangan Inti Oosit Sapi Setelah 24 Jam Presevasi Ovarium

Jurnal Agripet ◽

10.17969/agripet.v15i2.2417 ◽

2015 ◽

Vol 15 (2) ◽

pp. 72-78

Author(s):

Rini Widyastuti ◽

Siti Darodjah Rasad

Keyword(s):

Phosphate Buffer ◽

Bovine Oocyte ◽

Oocyte Maturity ◽

Phosphate Buffer Saline ◽

Bovine Oocytes ◽

Meiotic Competence

(Nuclear maturity of bovine oocyte after 24 hours ovary preservation)ABSTRACT. The objective of the research was to investigate their meiotic competence or nuclear maturity of bovine oocytes maturity in vitro after 24 hour preservation on 5°C. Oocytes were collected by slicing the ovaries in modified phosphate buffer saline (m-PBS). Selected cumulus-oocyte complexes (COCs) homogenous ooplasm were cultured in maturity medium at 38°C in humidified atmosphere of 5% CO2 incubator. After 24 hours, oocytes stained for nuclear maturity’s evaluation. The proportion of oocytes at metaphase II (MII) was significantly difference (P 0.05) on oocytes that 24 hours preservated (44.21 ± 3.04%) vs oocytes from fresh ovary (73.97 ± 9.32%) (P0.05). These results indicated that 24 hour’s preservation bovine’s ovary on 50C cause decreases of nuclear oocyte maturity.

Download Full-text