146. MOLECULAR FILTRATION PROPERTIES OF THE EXPANDED CUMULUS MATRIX: CONTROLLED SUPPLY OF METABOLITES AND EXTRACELLULAR SIGNALS TO CUMULUS CELLS AND THE OOCYTE

2010 ◽  
Vol 22 (9) ◽  
pp. 64
Author(s):  
K. R. Dunning ◽  
L. N. Watson ◽  
J. G. Thompson ◽  
R. L. Robker ◽  
D. L. Russell
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Oocyte Competence ◽  
Signalling Molecules ◽  
The Matrix ◽  
Filtration Properties

Cumulus matrix genes are positively correlated with oocyte competence [1]. Formation of the expanded cumulus matrix during oocyte maturation is well described; however its function remains elusive. We investigated whether cumulus matrix acts as a molecular filter, based on recognised filtration properties of analogous matrices. We found that cumulus matrix controls metabolite supply to the oocyte and retains prostaglandin E2 (PGE2), which is critical in oocyte maturation. The uptake of fluorescently labelled hydrophilic and hydrophobic metabolites showed that cumulus matrix formation significantly impeded diffusion to the oocyte. Expanded in vivo matured cumulus oocyte complexes (COCs, eCG+hCG16h) resisted uptake of glucose and cholesterol compared to unexpanded (eCG44h, P < 0.05), as assessed by confocal microscopy and spatial quantitation of fluorescence (P < 0.05). In vitro maturation (IVM) results in pronounced compositional deficiency of cumulus matrix proteins [2] and poor oocyte quality. Glucose and cholesterol were transported more readily into cumulus cells and the oocyte of IVM COCs (matured in αMEM/5% FCS/50 mIU/mL FSH, 16 h) compared to in vivo matured COCs (P < 0.05 and P = 0.08, respectively). Taking the inverse approach we found that PGE2 synthesised by cumulus cells is retained within the matrix compartment of in vivo matured COCs but IVM COCs did not retain PGE2 and secreted 4.3-fold more into the media. The relationship of retained to secreted PGE2 was significantly higher after in vivo maturation vs IVM COCs (P < 0.0001). This property of the COC matrix reveals a potential mechanism whereby the prostaglandin signal intensifies through a physicochemical mechanism rather than gene regulation. This is the first demonstration that cumulus matrix regulates diffusion toward and secretion from the COC, thus excluding glucose, known to negatively affect oocyte quality, and trapping factors, including PGE2, with critical roles in oocyte maturation and fertilisation. Thus, IVM may reduce oocyte quality due to poor trafficking of metabolites and signalling molecules. (1) McKenzie LJ, et al. Human cumulus granulosa cell gene expression: a predictor of fertilization and embryo selection in women undergoing IVF. Hum Reprod 2004; 19: 2869–2874.(2) Dunning KR, et al. Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes. Hum Reprod 2007; 22: 2842–2850.

188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

2017 ◽  
Vol 29 (1) ◽  
pp. 202 ◽  
Author(s):  
A. Lange-Consiglio ◽  
C. Perrini ◽  
P. Esposti ◽  
F. Cremonesi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Chi Square ◽  
Cell Layers ◽  
Hoechst Staining

The in vitro maturation of canine oocyte is problematic because it is difficult to reproduce the oviducal microenvironment where the in vivo maturation occurs. Because cells are able to communicate with each other by paracrine action, oviducal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MV), which represent a carrier for nonsoluble molecules including microRNA. The aim of the present work was to investigate the effect of the addition of CM or MV, secreted by oviducal cells, to the canine in vitro maturation medium. To generate CM, cells from oviducts of 3 animals in late oestrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500 × g, and stored at −80°C. Microvesicles were obtained by ultracentrifugation of CM at 100,000 × g for 1 h at 4°C and measured for concentration and size by a Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the oestrous cycle. Cumulus-oocyte complexes were released by slicing the ovarian cortex with a scalpel blade, and only Grade 1 cumulus-oocyte complexes (darkly granulated cytoplasm and surrounded by 3 or more compact cumulus cell layers) 110 to 120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24 h in SOF with 5.0 μg mL−1 of LH followed by 48 h in SOF supplemented with 10% of oestrous bitch serum and 10% CM or 50, 75, 100, or 150 × 106 MV mL−1 labelled with PKH-26. Control was the same medium without CM or MV. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII), by Hoechst staining, and the incorporation of MV. Statistical analysis was performed by chi-square test. Results show that canine oviducal cells secreted MV of 234 ± 23 nm in size, underling that these MV fall within the shedding vesicles category. The incorporation of labelled MV occurred at first in cumulus cells, at 48 h of maturation, and then, at 72 h, in oocyte cytoplasm. These MV had a positive effect on maturation rate (MII) at the concentration of 75 and 100 × 106 MV mL−1 compared with CM and control (20.34 and 21.82 v. 9.09 and 3.95%, respectively). The concentration of 150 × 106 MV mL−1 provided only 9.26% of MII. To understand the role of MV, we assessed the expression of 3 microRNA (miRNA-30b, miR-375, and miR-503) that are involved in some key pathways (WNT, MAPK, ERbB, and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MV is possibly due to the high level of miR-375, which recent literature shows to suppress the TGFβ pathway, leading to impaired oocyte maturation. In conclusion, the oviducal MV, or specific microRNA, are involved in cellular trafficking during oocyte maturation, and their possible use in vitro could facilitate the exploitation of canine reproductive biotechnologies.

Effect of butafosfan supplementation during oocyte maturation on bovine embryo development

Zygote ◽  
2019 ◽  
Vol 27 (05) ◽  
pp. 321-328
Author(s):  
Lucas Teixeira Hax ◽  
Joao Alveiro Alvarado Rincón ◽  
Augusto Schneider ◽  
Lígia Margareth Cantarelli Pegoraro ◽  
Letícia Franco Collares ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Nuclear Maturation

SummaryAround 60–80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus–oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

scholarly journals Localisation of the hyaluronan receptor CD44 in porcine cumulus cells during in vivo and in vitro maturation

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 317-326 ◽  
Author(s):  
Masaki Yokoo ◽  
Paisan Tienthai ◽  
Naoko Kimura ◽  
Koji Niwa ◽  
Eimei Sato ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Binding Ability ◽  
Migration Ability ◽  
And Migration ◽  
Boar Spermatozoa

Polyspermy is fairly common during porcine in vitro fertilisation (IVF), perhaps due to incomplete in vitro oocyte maturation (IVM). Porcine cumulus cells (CCs) layered around the oocyte produce large amounts of extracellular hyaluronan (HA) when forming an expanding cell cloud during the last phase of oocyte maturation. The specific actions of HA are mediated via HA-binding proteins (HABPs), such as CD44, which act as receptors. In this study using immunocytochemistry and western blotting we investigated the localisation of CD44 in CCs obtained from in vivo-matured pig cumulus-oocyte complexes (COCs) and compared it with that in CCs from immature COCs and of COCs subjected to IVM and IVF procedures. Immunolabelling of CD44 was absent or very weak in CCs from immature COCs but strongly present on the surface of the CCs obtained from in vivo, displaying a similar localisation in the in vitro-matured COCs. In the latter, the labelling decreased but did not disappear in CCs 4 h after sperm co-incubation during IVF. Immunoblotting detected bands of between 73 and 88 kDa, corresponding to CD44, in the protein extract from in vivo CCs collected immediately prior to, or following spontaneous ovulation. The in vitro-matured CCs, however, presented bands ranging from 81 kDa to 88 kDa. Also, the bands found in the in vivo-matured CCs showed a larger variation of intensity and migration among animals than did the batches of in vitro-matured CCs. No CD44 band was detected on aliquots of the frozen-thawed boar spermatozoa used for IVF. The results clearly demonstrate that the specific HA receptor CD44 is present in expanding CCs of in vivo-matured pig COCs, in relation to increasing amounts of inter-CC HA. The subtle differences in molecular weight and migration ability observed between in vivo and in vitro samples may relate to differences in glycosylation and thus explain differences in HA-binding ability, of consequence for optimising in vitro culture conditions.

157 EFFECT OF FSH STARVATION (COASTING) FOLLOWING SUPEROVULATION ON OOCYTE COMPETENCE AND CLONING EFFICIENCY IN GOATS

2017 ◽  
Vol 29 (1) ◽  
pp. 187 ◽  
Author(s):  
C. E. Méndez-Calderón ◽  
C. R. Lazzarotto ◽  
L. H. Aguiar ◽  
F. L. Ongaratto ◽  
K. C. S. Tavares ◽  
...  
Keyword(s):  
Pregnancy Outcome ◽  
In Vitro Maturation ◽  
Animal Health ◽  
Oocyte Quality ◽  
Control Group ◽  
In Vitro Production ◽  
Oocyte Competence ◽  
Maturation Rate

Oocyte competence plays a key role in the overall efficiency of reproductive biotechnologies. In cattle, FSH starvation following superovulation (coasting) improves oocyte competence, blastocyst yield and pregnancy outcome when used in ovum pickup-in vitro production programs. The aim of this study was to compare the effect of coasting after exogenous FSH stimulation on goat oocyte quality and competence to support in vitro maturation and in vivo embryo development following cloning procedures in goats. Donor and recipient preparation, cumulus-oocyte complex (COC) retrieval and selection, IVM, cloning by somatic cell nuclear transfer, embryo transfer, and pregnancy diagnosis (Days 23–26) were performed according to our established procedures [Martins et al. 2016 doi: 10.1089/cell.2015.0082]. Cumulus-oocyte complexes were obtained in vivo from 71 cycling FSH-stimulated mature Nubian-crossed goats, combined or not with FSH starvation (coasting period). Donor females were oestrous synchronized with a progesterone intravaginal insert (Day 0). On Day 10, a 0.75-mg D-cloprostenol dose was given IM, with the onset of the superovulation treatment, composed of five 20-mg FSH doses (Folltropin®, Bioniche Animal Health, Pullman, WA, USA), via IM at 12-h intervals. Donors were subjected to laparoscopic ovum pickup either 9 h (control group, n = 36) or 21 h (coasting group, n = 35) after the last FSH dose, respectively. Skin fibroblast cell cultures from a male neonate were co-transfected with a mammary gland expression vector with the human lactoferrin (hLF) coding sequence and with CRISPR/Cas9 system either for the PRNP prion gene or the Rosa26 locus. A bi-allelic hLF-PRNP and a mono-allelic hLF-Rosa26 cell colony were used for cloning. Data were compared by ANOVA or the χ2 test (P < 0.05). No differences were observed between control and coasting for number of follicles (18.7 ± 1.4 v. 21.2 ± 1.7), and retrieved (17.3 ± 1.2 v. 20.7 ± 1.9), viable (15.9 ± 1.1 v. 19.6 ± 1.8), Grade I (1.5 ± 0.3 v. 2.5 ± 0.5), and Grades III+IV (6.0 ± 0.6 v. 5.7 ± 0.7) COC, as well as for COC retrieval (92.4%, 574/621 v. 94.5%, 685/725) and fusion (62.8%, 273/435 v. 61.3%, 311/507) rates, respectively, irrespective of the cell lines. However, the coasting group rendered higher number of Grade II COC (11.3 ± 1.2 v. 8.4 ± 0.7), number and proportion of Grades I+II COC (13.9 ± 1.5 v. 9.9 ± 0.9, 70.8% v. 62.4%), and maturation rate (70.9% v. 65.4%) than the control group, respectively, for a lower proportion of Grades III+IV (29.2% v. 37.6%, respectively). A total of 213 and 233 Day-1 cloned embryos from the control and the coasting groups were transferred to 18 (96/9 hLF-PRNP and 117/9 hLF-Rosa26 cells) and 19 (128/11 hLF-PRNP and 105/8 hLF-Rosa26 cells) female recipients, respectively, resulting in 1/9 (11.1%) and 4/11 (36.4%) pregnancies from the hLF-PRNP cells, and 3/9 (33.3%) and 3/8 (37.5%) from the hLF-Rosa26 cells, for the control (4/18, 22.2%) and coasting (7/19, 36.8%) groups, respectively, for an overall pregnancy rate of 29.7% (11/37). In conclusion, the use of coasting improved oocyte quality and in vitro maturation rate, also appearing to increase pregnancy outcome after goat cloning.

In vivo and in vitro effects of interleukin-1β on equine oocyte maturation and on steroidogenesis and prostaglandin synthesis in granulosa and cumulus cells

2009 ◽  
Vol 21 (2) ◽  
pp. 265 ◽  
Author(s):  
Maud Caillaud ◽  
Nadine Gérard
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Interleukin 1Β ◽  
Cox 2 ◽  
Group 2 ◽  
Cox 1

We analysed the effect of interleukin-1 on oocyte maturation and on steroid and prostaglandin production by equine granulosa and cumulus cells. In Experiment 1, interleukin-1β (IL-1β) was injected into the growing dominant follicle, which was punctured 38 h later. Follicular fluid was assayed for steroids and prostaglandin-F2α (PGF2α). Granulosa cells were analysed for 3β-hydroxysteroid dehydrogenase (3β-HSD), progesterone receptor (PR), cyclooxygenase 1 and 2 (Cox 1 and Cox 2) and steroidogenic acute regulatory protein (StAR) mRNAs. In Experiment 2, cumulus–oocyte complexes (COCs) were collected from slaughterhouse ovaries and cultured in different media: control group (TCM199 + BSA); Group 2 (+ IL-1β); Group 3 (+ EGF); Group 4 (+ EGF + IL-1β); and Group 5 (+ EGF + IL-1β + IL-1RA). Cumulus cells were analysed for 3β-HSD, PR, Cox 1, Cox 2 and StAR mRNAs. After injections of crude equine gonadotropin (CEG; LH effect) or IL-1β, progesterone and PGF2α levels increased, whereas 17β-oestradiol decreased. EGF induced an increase in the rate of in vitro maturation (P < 0.05), whereas IL-1β had a limited effect. IL-1β significantly decreased the rate of EGF-induced oocyte maturation (P < 0.05). Cox 2 mRNA level increases in granulosa cells after CEG injection (P = 0.07). In cumulus cells, StAR and PR mRNAs were lower in Group 2 and 3β-HSD mRNA was higher in Groups 4 and 5. These data confirm that IL-1 is involved in equine oocyte in vitro maturation. We demonstrated in vivo that IL-1β has an effect on steroids and PGF2α secretion in the preovulatory follicle.

scholarly journals Investigating the Intrafollicular Factors Affecting Oocyte Developmental Competency

2021 ◽  
Author(s):  
◽  
Zaramasina Clark
Keyword(s):  
Gene Expression ◽  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Embryo Quality ◽  
Cumulus Cells ◽  
Significant Finding ◽  
High Quality ◽  
Final Maturation

<p>The number of cycles of assisted reproductive technologies (ART) performed increased by ~9.5 % globally between 2008 and 2010. In spite of this, the success rate in terms of delivery was only ~19.0 % (Dyer et al., 2016). This discrepancy between the demand for, and success of, these technologies necessitates the development of tools to improve ART efficiency. To facilitate this, a better understanding of how the microenvironment changes within the developing follicle to culminate in a mature, developmentally-competent oocyte is required. This study employed an in vivo and in vitro ovine model to investigate the relationship between the surrounding microenvironment and oocyte maturation, and in particular, the attainment of oocyte developmental competency and high-quality embryos.  The first objective of this PhD study was to comprehensively investigate the changing microenvironment of in vivo matured, presumptive preovulatory (PPOV) follicles from wild-type (++) and high ovulation rate (OR; I+B+) ewes. The high OR ewes were heterozygous carriers of mutations in BMP15 (I+) and BMPRIB (B+). Functional differences in follicular somatic (granulosa and cumulus) cells between these genotypes, including differential gonadotropin responsiveness of granulosa cells, composition of follicular fluid and gene expression profiles in cumulus cells were evident. These differences emerged as part of a compensatory mechanism by which oocytes from smaller follicles, containing fewer granulosa cells, achieved developmental competency in I+B+ ewes.  The second objective of this PhD study was to develop new approaches for improving current in vitro maturation (IVM) strategies. The first approach utilised in this study focused on developing biomarkers that could be used to improve prediction of developmental competency in oocytes and in vitro produced embryos. This involved interrogating the hypothesis that a combination of molecular and morphokinetic biomarkers would better predict the developmental competency of oocytes and embryos compared to using these biomarkers alone. The second approach utilised in this PhD study tested the effects of modulating IVM conditions to better mimic the follicular microenvironment of a high, compared to a low, OR species on oocyte developmental competency and embryo quality. This involved supplementing IVM media with different ratios of two oocyte-secreted growth factors, i.e. GDF9:BMP15, that were representative of low or high OR species. These approaches demonstrated significant potential and warrant further investigation.  The most significant finding of this study was that despite variances in the surrounding microenvironment during in vivo and in vitro oocyte maturation that culminated in differential gene expression patterns in cumulus cells, and divergent gonadotropin-responsiveness of granulosa cells, the gene expression signatures of developmentally-competent oocytes and the morphokinetics of high-quality embryos were unaltered. This confirms the value of developing such biomarkers for oocyte development competency and embryo quality that remain unaltered despite a changing surrounding environment. Interestingly, simulating the ratio of GDF9:BMP15 that oocytes from high OR species are exposed to during maturation improved developmental competency in oocytes as demonstrated by increased blastocyst rates. Furthermore, this study has demonstrated that combinations of molecular (cumulus cell gene expression) and morphokinetic biomarkers improved the ability to predict developmental competency in oocytes and embryos. Overall, this study revealed novel information regarding the follicular microenvironment during final maturation and identified several novel approaches to improving the efficiency of ART.</p>

P–442 Cancer does not adversely affect oocyte maturation in vitro with the exception of breast cancer

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
I Viran. . Klun ◽  
J Bedenk ◽  
N Jancar
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Hormonal Stimulation ◽  
Infertile Women ◽  
Maturation In Vitro ◽  
Infertile Patients

Abstract Study question Do different types of cancer affect the success of oocyte maturation in vitro compared to infertile women included in the in vitro fertilization (IVF) program? Summary answer Cancer does not adversely affect oocyte maturation in vitro, with the exception of breast cancer, compared to infertile women in the in vitro fertilization program. What is known already Vitrification and storage of oocytes in liquid nitrogen is one of the real options for maintaining reproductive function in cancer patients. Despite careful hormonal stimulation of the ovaries, however, the proportion of oocytes is immature and lost to the patient. In vitro maturation of oocytes can play an important role in resolving immature oocytes and increasing the chances of conception in cancer patients. Moreover, it can mean a safe way to store oocytes when ovarian hormonal stimulation could worsen the disease. Therefore, the aim of this study was to determine whether different types of cancer affect oocyte in vitro maturation. Study design, size, duration After ovarian stimulation in 18 cancer patients, the number and maturity of oocytes were compared to 21 infertile patients in the IVF program over a three-year period. In both groups, 119 germinal vesicle-GV oocytes were matured in vitro to compare the maturation rate. After IVF in a subset of 17 infertile patients, the fertilization of in vitro and in vivo matured oocytes was compared in the same cycles. The procedure was considered in cancer patients. Participants/materials, setting, methods In this prospective study, forty-five GV oocytes in cancer patients and 74 GV oocytes in infertile patients underwent in vitro maturation procedure. Each oocyte was matured in vitro in the MediCult IVM System by conditioning in LAG medium and maturation for up to 28 hours in IVM medium with added hormones FSH and hCG, in coculture with cumulus cells from mature oocytes in the same patients. Oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Main results and the role of chance After controlled ovarian hormonal stimulation, 198 oocytes were retrieved in cancer patients and 259 oocytes in infertile women and there were no significant differences in the number of retrieved oocytes, proportion of degenerated oocytes and proportion of GV oocytes. In cancer patients, the proportion of oocytes that matured in vitro was lower than in infertile patients (66.0 vs. 80.0%), but the difference was not significant. Among cancer patients, the oocyte maturation rate tended to be lower in patients with breast cancer than in patients with other cancers (54.5% vs. 81.2%; difference not significant). However, in patients with breast cancer, significantly fewer oocytes matured in vitro than in infertile patients (54.5% vs. 80.0%; P &lt; 0.05, Chi-Square test) even though they tended to be younger (29.3 ± 7.4 vs. 33.4 ± 5.0 years; non-significant difference). After in vitro maturation, there was a 13% increase in mature oocyte yield in cancer patients and a 20.1% increase in infertile women with no significant difference observed. After ICSI in a subset of infertile women, there was approximately the same fertilization rate between oocytes matured in vitro and in vivo (55.1% vs. 57.0%) in the same cycles. Limitations, reasons for caution For ICSI in oocytes matured in vitro, we had to use semen collected the day before, while oocytes matured in vivo were fertilized with fresh semen in the same cycle. Therefore, we could not compare the development of embryos in both groups. Wider implications of the findings: In vitro maturation of oocytes in connection with their vitrification or vitrification of embryos after their fertilization appears to be a valuable way to maintain the fertility of young cancer patients, but a worse outcome is expected in breast cancer patients. Trial registration number National Medical Ethical Committee Approval, No. 0120–222/2016–2; KME 115/04/16.

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

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.

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 260
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Cell Morphology ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Oocyte Developmental Competence ◽  
Before And After ◽  
Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

172 DETERMINING THE REQUIREMENTS FOR LH AND FSH DURING SHEEP IN VITRO OOCYTE MATURATION

2014 ◽  
Vol 26 (1) ◽  
pp. 200 ◽  
Author(s):  
C. de Frutos ◽  
R. Vicente-Perez ◽  
P. J. Ross
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Mixed Logit ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Developmental Competence ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Grade 3

In vitro maturation (IVM) of oocytes in domestic animals is a widespread practice of research and commercial relevance. Gonadotropic hormones are typically supplemented to the IVM medium to stimulate resumption of meiosis, progression to metaphase II (MII), and oocyte developmental competence. The common use of pituitary-derived products presents 2 problems: contamination from other pituitary hormones and inconsistences from batch-to-batch variation. Recombinant hormones can help circumvent these issues and identify specific gonadotropin requirements for in vitro maturation. The aim of the present study was to determine the effect of supplementing recombinant bovine LH and/or FSH (AspenBio) to the maturation of ovine oocytes in terms of cumulus expansion and progression to the MII stage. Abattoir-derived sheep cumulus–oocyte complexes (COC) were obtained from 1- to 5-mm-diameter antral follicles by ovary slicing. Oocytes with a homogeneous cytoplasm surrounded by at least 3 layers of cumulus cells were selected and cultured in serum-free IVM medium (Cotterill et al. 2012 Reproduction 144, 195–207) at 38.5°C and 5% CO2. The COC obtained from 8 replicates were allocated into 4 experimental groups: (1) no hormones; (2) 1.5 μg mL–1 recombinant bovine LH (rbLH); (3) 1.5 μg mL–1 recombinant bovine FSH (rbFSH); and (4) rbLH and rbFSH. The expansion of cumulus cells was recorded in each group after 24 h of IVM and COC classified as (1) very poor or no cumulus expansion (grade 1); (2) limited cumulus expansion (grade 2); and (3) full cumulus expansion (grade 3). Nuclear maturation in the 4 treatments was evaluated by assessing progression to the MII stage via DNA staining with Hoechst 33342 and fluorescence imaging. The effect of treatment on the observed proportion of MII oocytes was evaluated using a mixed logit model including treatment and replicate as fixed and random effects, respectively. Culture in IVM medium in the absence of gonadotropins or in the presence of rbLH resulted in poor cumulus expansion (grade 1). The supplementation of IVM medium with rbFSH (with or without rbLH) yielded a high degree of cumulus expansion (grades 2–3). Likewise, addition of rbFSH enhanced progression of oocytes to the MII stage, whereas use of rbLH, although it had an effect on progression to MII, did not augment the effect of rbFSH (Table 1). These results indicate that rbFSH is necessary and sufficient to induce sheep oocyte maturation in a high proportion of oocytes. Table 1.Cumulus expansion and oocyte nuclear stage after IVM

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