The new system of shorter porcine oocyte in vitro maturation (18 hours) using ≥8 mm follicles derived from cumulus-oocyte complexes

Despite recent efforts to improve in vitro maturation (IVM) systems for porcine oocytes, developmental competence of in vitro-matured oocytes is still suboptimal compared with those matured in vivo. In this study, we compared oocytes obtained from large (≥8 mm; LF) and medium (3–7 mm; MF) sized follicles in terms of nuclear maturation, intracellular glutathione and reactive oxygen species levels, gene expression, and embryo developmental competence after IVM. In the control group, cumulus-oocyte complexes (COCs) were aspirated from MF and matured for 22 hours with hormones and subsequently matured for 18 to 20 hours without hormones at 39 °C, 5% CO₂in vitro. In the LF group, COCs were obtained from follicles larger than 8 mm and were subjected to IVM for only 18 hours. The ovaries have LF were averagely obtained with 1.7% per day during 2012 and it was significantly higher in the winter season. The results of the nuclear stage assessment of the COCs from the LFs are as follows: before IVM (0 hours); germinal vesicle stage (15.2%), metaphase I (MI) stage (55.4%), anaphase and telophase I stages (15.8%), and metaphase II (MII) stage (13.6%). After 6 hours IVM; germinal vesicle (4.2%), MI (43.6%), anaphase and telophase I (9.4%), and MII (42.8%). After 18-hour IVM; MI (9.7%) and MII (90.3%). Oocytes from LF showed a significant (P < 0.001) increase in intracellular glutathione (1.41 vs. 1.00) and decrease in reactive oxygen species (0.8 vs. 1.0) levels compared with the control. The cumulus cells derived from LFs showed lower (P < 0.1) mRNA expression of COX-2 and TNFAIP6, and higher (P < 0.1) mRNA expression of PCNA and Nrf2 compared with the control group-derived cumulus cells. After parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (SCNT) using matured oocytes from LFs, the embryo development was significantly improved (greater blastocyst formation rates and total cell numbers in blastocysts) compared with the control group. In conclusion, oocytes from LFs require only 18 hours to complete oocyte maturation in vitro and their developmental competence is significantly greater than those obtained from MFs. Although their numbers are limited, oocytes from LFs might offer an alternative source for the efficient production of transgenic pigs using SCNT.     

Developmental capability of denuded bovine oocyte in a co-culture system with intact cumulus-oocyte complexes: role of cumulus cells, cyclic adenosine 3',5'-monophosphate, and glutathione

Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte-sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co-cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus-oocyte complexes (COCs) in co-culture with oocytes freed of CC investment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3'-5' monophosphate (cAMP) and glutathione (GSH) during FSH-stimulated maturation of denuded oocyte co-cultured with intact COCs. Our data confirm that denuded oocyte has a scarce developmental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co-presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic developmental potential is achieved only when DOs are co-cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothesize that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete.     

Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors

Background

Single embryo transfer (SET) is the most successful way to reduce the frequency of multiple pregnancies following in vitro fertilisation. However, selecting the embryo for SET with the highest chances of pregnancy remains a difficult challenge since morphological and kinetics criteria provide poor prediction of both developmental and implantation ability. Partly through the expression of specific genes, the oocyte-cumulus interaction helps the oocyte to acquire its developmental competence. Our aim was therefore to identify at the level of cumulus cells (CCs) genes related to oocyte developmental competence.

Methodology/Principal Findings

197 individual CCs were collected from 106 patients undergoing an intra-cytoplasmic sperm injection procedure. Gene expression of CCs was studied using microarray according to the nuclear maturity of the oocyte (immature vs. mature oocyte) and to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilisation). Microarray study was followed by a meta-analysis of the behaviour of these genes in other datasets available in Gene Expression Omnibus which showed the consistency of this list of genes. Finally, 8 genes were selected according to oocyte developmental competence from the 308 differentially expressed genes (p<0.0001) for further validation by quantitative PCR (qPCR). Three of these 8 selected genes were validated as potential biomarkers (PLIN2, RGS2 and ANG). Experimental factors such as inter-patient and qPCR series variability were then assessed using the Generalised Linear Mixed Model procedure, and only the expression level of RGS2 was confirmed to be related to oocyte developmental competence. The link between biomarkers and pregnancy was finally evaluated and level of RGS2 expression was also correlated with clinical pregnancy.

Conclusion/Significance

RGS2, known as a regulator of G protein signalling, was the only gene among our 8 selected candidates biomarkers of oocyte competence to cover many factors of variability, including inter-patient factors and experimental conditions.     

BIM EL-mediated apoptosis in cumulus cells contributes to degenerative changes in aged porcine oocytes via a paracrine action

Whether cumulus cells (CCs) contribute to oocyte aging remains controversial; in that regard, little is known about biochemical processes of gene expression in CCs surrounding aged oocytes. The objective was to elucidate contributions of CCs to porcine oocyte aging and degeneration, apoptosis and BIM expression in CCs during oocyte aging in vitro. When culture of cumulus oocyte complexes (COCs) was prolonged (68 h, which resulted in 24 h of aging), the rate of blastocyst formation following electro-activation was lower than that of oocytes aged without CCs (2.6 ± 0.1 vs 13.5 ± 1.3%, mean ± SEM; P < 0.05). In addition, the presence of CCs significantly accelerated spontaneous fragmentation of oocytes following prolonged (92 h) culture. Apoptotic CCs were present in COCs cultured for 68 h, and the abundance of Bim mRNA in CCs progressively increased after 56 h of culture (P < 0.05). Based on immunofluorescence, BIM protein expression was up-regulated in CCs surrounding aged oocytes; furthermore, quantification (Western blot) of BIM_EL protein progressively increased after 56 h of culture. Lastly, in a series of experiments to elucidate the signal pathway, blocking gap junctions (with 1-octanol) during aging did not eliminate the effect of CCs on accelerating oocyte aging, but prolonged co-culture of denuded oocytes with COCs after in vitro maturation reduced blastocyst rate relative to culture of denuded oocytes aged alone (4.15 ± 0.1 vs 11.0 ± 0.7%, P < 0.05). We concluded that apoptotic CCs, in which BIM_EL up-regulation was involved, accelerated oocyte aging and degeneration in vitro via a paracrine action.     

The effects of season and moderate nutritional restriction on ovarian function and oocyte nuclear maturation in cycling gilts

The fertility of female pigs is impaired during summer and in response to restriction of feed intake, resulting in reduced productivity of the breeding herd. This study determined the effect of season and moderate nutritional restriction on ovarian function and oocyte developmental competence of cycling gilts. Eighty prepubescent gilts were used across two seasons—summer (S: January to March) and winter (W: June to August)—and received either a high (2.5× maintenance) or a moderately restricted (1.5× maintenance) feeding level for the first 19 days of their second estrous cycle. On Day 19, ovaries were collected post-slaughter. Diameters of all surface follicles over 1 mm were measured. All follicles ≥4 mm were aspirated and cumulus–oocyte complexes underwent in vitro maturation for ∼44 hours to assess oocyte developmental competence on the basis of metaphase II (MII) attainment. Moderate dietary nutrition reduced daily liveweight gain but did not affect the ovarian follicle population or oocyte developmental competence. The number of large follicles (≥6 mm) was lower during summer (S: 10.7 ± 1.74 vs. W: 15.5 ± 1.15, P < 0.05), as was the proportion of oocytes at the germinal vesicle stage of meiosis (S: 0.06 ± 0.02 vs. W: 0.08 ± 0.02, P < 0.05). However, the proportion of oocytes attaining MII was similar in summer and winter (S: 0.72 ± 0.04 and W: 0.69 ± 0.06, P > 0.05). Intrafollicular concentrations of luteinizing hormone were higher in summer (S: 43.05 ± 6.44 vs. W: 12.05 ± 5.12 ng/mL, P < 0.001), whereas estradiol was lower (S: 1.27 ± 0.36 vs. W: 27.52 ± 5.59 ng/mL, P < 0.001). In conclusion, our data demonstrated that in summer, follicle growth beyond 6 mm is impaired during the periovulatory period, without affecting oocyte meiotic competence. Importantly, these data also demonstrated that ovarian follicle growth and the capacity of oocytes to reach MII in vitro appear unaffected by moderate nutritional restriction during the preceding estrous cycle.     

Reduction of nitric oxide level results in maturation promoting factor destabilization during spontaneous meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro

Nitric oxides (NO) act as one of the major signal molecules and modulate various cell functions including oocyte meiosis in mammals. The present study was designed to investigate the mechanism of NO action during spontaneous meiotic exit from diplotene arrest (EDA) in rat cumulus oocytes complexes (COCs) cultured in vitro. Diplotene‐arrested COCs collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h were exposed to various concentrations of NO donor, S‐nitroso‐N‐acetyl penicillamine (SNAP) and inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG) for 3 h in vitro and downstream factors were analyzed. Our results suggest that SNAP inhibited, while AG induced EDA in a concentration‐dependent manner. The iNOS‐mediated total NO, cyclic nucleotides and cell division cycle 25B (Cdc25B) levels were reduced significantly. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated cyclin‐dependent kinase 1 (Cdk1) level and decreased Thr161 phosphorylated Cdk1 as well as cyclin B1 levels leading to maturation promoting factor (MPF) destabilization. The destabilized MPF finally induced spontaneous EDA. Taken together, these results suggest that reduction of iNOS‐mediated NO level destabilizes MPF during spontaneous EDA in rat COCs cultured in vitro.     

Effect of different cryo-devices on in vitro maturation and development of vitrified-warmed immature buffalo oocytes

The aim of the study was to identify a cryo-device that would be best suited for the vitrification of buffalo immature cumulus-oocyte complexes (COCs) as judged by viability and meiotic competence of the vitrified-warmed oocytes and their development ability following in vitro fertilization (IVF). The expression of oocyte secreting factors and their receptors (GDF9, BMP15, BMPR2, TGFBR1) and apoptosis related genes (BCL2, BAX, P53, C-MYC) were compared in vitrified-warmed oocytes after in vitro maturation. COCs from the ovaries of slaughtered buffaloes were vitrified in a combination of dimethyl sulfoxide, ethylene glycol, and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). The fresh COCs were exposed to vitrification and warming solutions as in other vitrification methods without plunging in to liquid nitrogen (EC). The viability of vitrified-warmed COCs, 2 h post warming in HS and CT was similar to fresh and EC groups but significantly higher than CS and OPS methods. The proportions of oocytes with first polar body after 24 h in vitro maturation were significantly higher in HS and CT methods than in CS, OPS and CL methods. The development ability of these vitrified-warmed oocytes to blastocyst stage following IVF in all vitrified groups was significantly lower than control and EC groups. Among the vitrified groups, the blastocyst rate in HS, CT and CL groups was significantly higher than in OPS and CS groups. It was also observed that the expression levels of GDF9, BMP15, BMPR2, TGFBR1, BCL2, BAX, P53 and C-MYC genes in vitrified-warmed COCs in CT, HS and CL groups were similar to control. The results indicated that HS, CT and CL are more suitable cryo-devices for vitrification of buffalo immature oocytes.     

Follicular 17β-estradiol and progesterone concentrations and degree of cumulus cell expansion as predictors of in vivo-matured oocyte developmental competence in superstimulated heifers

The quality of an oocyte is crucial for successful generation of offspring, but few selection parameters have been identified that reliably predict oocyte developmental competence. The objective of the present study was to determine whether the developmental competence of in vivo-matured oocytes derived from superstimulated heifers could be predicted by 17β-estradiol and progesterone concentrations in follicular fluid, degree of cumulus cell expansion, and follicular diameter. Cumulus oocyte complexes were individually collected from follicles ≥8 mm 22 hours after an induced LH peak and individually fertilized and cultured in vitro. Only oocytes that originated from follicles with 17β-estradiol ≤0.25 μM and progesterone ≥0.26 μM developed into blastocysts. When a combination of these cutoff values was evaluated as a predictor of oocyte competence, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 75%, 49%, and 100%, respectively. Hormone concentrations in follicular fluid were also associated with the degree of cumulus cell expansion and only cumulus oocyte complexes with full expansion developed into blastocysts; sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 71%, 45%, and 100%, respectively, when full expansion was used as the predictive criterion for blastocyst production. Follicular diameter was not a good predictor of oocyte competence. In conclusion, concentrations of 17β-estradiol and progesterone in the preovulatory follicle and the degree of cumulus cell expansion are predictors of blastocyst production in superstimulated heifers and can be used as selection markers for oocyte developmental competency.     

Effect of follicular size on meiotic and developmental competence of porcine oocytes

In several species, the developmental competence of the oocyte is acquired progressively during late follicular growth, after the acquisition of the competence to resume and complete meiosis. In the pig, full meiotic competence of the oocyte is reached in ovarian follicles with a diameter of 3 mm or more. However, there is no information about developmental competence acquisition. We analyzed the ability of oocytes from three foll icular size classes to resume and complete meiosis, to be fertilized, and to develop in vitro to the blastocyst stage. A total of 941 follicles were dissected from slaughterhouse gilt ovaries and classified as small (<3 mm, n = 330), medium (3-5 mm, n = 373), or large (>5 mm, n = 238). The cumulus-oocyte complexes recovered from these follicles were submitted to in vitro maturation for 44 h in TCM199 supplemented with 10 ng/ml EGF, 400 ng/ml pFSH and 570 microM cysteamine; in vitro fertilized for 18 h in mTBM with 10(5) frozen-thawed percoll-selected sperms/ml; and developed for 7 days in mSOF. Samples of oocytes or presumptive zygotes were fixed and stained at the end of maturation and fertilization. Groups of oocytes were cultured for 3 h in the presence of 35S-methionine before or after maturation for SDS-PAGE analysis of protein neosynthesis. More oocytes originating from medium and large follicles were competent for maturation than oocytes from small follicles (77 and 86% of metaphase II, respectively, versus 44%, P < 0.05). More oocytes from medium and large follicles werepenetratedby spermatozoa during in vitro fertilization, resulting in significantly more oocytes presenting two or more pronuclei at the end of fertilization (73 and 77% for medium and large follicles, respectively, versus 53% for small follicles, P < 0.05). More oocytes from medium and large follicles developed to the blastocyst stage (14 and 23%, respectively) than those from small follicles (3%, P < 0.05), even if the development rates were corrected by the maturation or fertilization rates. It is concluded that a high proportion of oocytes harvested from follicles of less than 3 mm in the pig are not fully competent for meiosis and are cytoplasmically deficient for development.     

Connexin 43 coupling in bovine cumulus cells,during the follicular growth phase,and its relationship to in vitro embryo outcomes

Gap junctional coupling between cumulus cells is required for oocytes to reach developmental competence. Multiple connexins, which form these gap junctions, have been found within the ovarian follicles of several species including bovine. The aim of this study was to determine the role of connexin 43 (CX43) and its relationship to embryo development, after in vitro fertilization (IVF). Cumulus?oocyte complexes (COCs) were obtained from abattoir sourced, mixed breed, bovine ovaries. COCs were isolated from follicles ranging from 2 to 5 mm in size, representing the preselected follicle pool. Immediately after isolation, two cumulus cell biopsies were collected and stored for analysis pending determination of developmental outcomes. Using in vitro procedures, COCs were individually matured, fertilized, and cultured to the blastocyst stage. Biopsies were grouped as originating from COCs that arrested at the two‐cell stage (low developmental competence [LDC]) or having developed to the late morula/blastocyst stage (high developmental competence [HDC]), after IVF and embryo culture. The expression level of CX43 was found to be significantly higher in cumulus cells from COCs that had an HDC when compared with those that had an LDC. Moreover, the gap junctional intercellular coupling rate was significantly higher in cumulus from COCs deemed to have an HDC. Significantly higher expression of the cumulus health markers luteinizing hormone receptor and cytochrome p450 19A1 was found in the cumulus originating from oocytes with HDC, suggesting that this system may provide a mechanism for noninvasively testing for oocyte health in preselected bovine follicles.     

Cumulus cells accelerate aging of mouse oocytes by secreting a soluble factor(s)

Control of oocyte aging in vitro is important for both human-assisted reproduction and animal embryo technologies because fertilization or artificial activation of aged oocytes results in abnormal development. Interactions between somatic and germ cells are also an important issue in current biological research. The role of cumulus cells (CCs) in maturation, ovulation, and fertilization of oocytes has been extensively studied, yet little is known about their role in oocyte aging. Although our previous study has shown that CCs accelerate the aging progression of mouse oocytes, the mechanism by which CCs accelerate oocyte aging is unknown. In this study, cumulus-denuded mouse oocytes (DOs) were co-cultured with cumulus-oocyte complexes (COCs) or CC monolayer or cultured in medium conditioned with these cells and changes in the susceptibility to activating stimuli and in MPF activity of oocytes were evaluated after different aging treatments. The results showed that culture with or in medium conditioned with COCs or CC monolayer promoted activation of DOs, indicating that a soluble factor is responsible for the aging-promoting effect. The in vivo and in vitro-matured DOs did not differ in responsiveness to the aging-promoting factor (APF). Heat shock did not accelerate oocyte aging unless in the presence of CCs. The production of APF was not affected by the age or maturation system of COCs, but increased with their density and duration of culture. The results strongly suggest that CCs accelerated oocyte aging by secreting a soluble APF into the medium. Further analysis showed that the APF was heat labile but stable to freezing, it had a threshold effective concentration and can be depleted by DOs.     

In vitro development of individually matured bovine oocytes in relation to follicular wall atresia

Morphologically good-quality cumulus oocyte complexes (COCs) can originate from slightly atretic follicles. Biochemical and ultrastructural investigations reveal that a very high percentage of bovine antral follicles express some degree of atresia. The aim of the present study was to determine the developmental competence of good quality COCs in relation to their biochemically estimated follicular wall apoptosis. For experimental design a single oocyte maturation system was established, followed by group culture processing oocytes together according to their level of follicular wall atresia estimated by an ELISA for apoptotic cell death. Single oocyte culture during maturation reduced the developmental capacity of oocytes significantly (P < 0.01), with 5% blastocysts versus 25% after common group culture. Blastocyst formation for single oocyte maturation was found exclusively in oocytes isolated from luteal stage ovaries with low degree of apoptosis. The level of follicular wall apoptosis in luteal stage follicles (0.79 +/- 0.05 units/mg protein, n = 198) was lower than in follicular stage follicles (1.14 +/- 0.05 units/mg protein, n = 208). This was caused by significant higher levels in small (< 3.5 mm diameter) and large (> 5.5 mm diameter) follicles of the latter group. In conclusion, despite reduced developmental capacity after single oocyte maturation, we were able to reveal some functional relationship between oocyte origin and quality. It was shown that morphologically good quality COCs isolated from follicles with higher degree of apoptosis lose their developmental capacity.     

Morphology and developmental competence of bovine oocytes relative to follicular status

In cattle, follicle dimension has been used as the main criterion for selection of oocytes for in vitro embryo production. However, follicles with similar diameters may be in very different physiologic phases. The aim of this study was to investigate whether morphology and developmental competence of cumulus-oocyte complexes (COCs) are related to the phase of development of the follicle, and presence of the corpus luteum (CL) or the dominant follicle in the ovary from which the COCs were collected. Cows (n = 143) were given a luteolytic dose of PGF(2alpha) and 8 days later underwent transvaginal ultrasound guided ablation of follicles > or =4mm to induce emergence of a new follicular wave. Cows (n = 10-20 per replicate) were slaughtered on Day 2, 3, 5 or 7 (Day 0 = follicular wave emergence), equivalent to the growing, early static, late static, and regressing phases of subordinate follicle development. COCs were collected from subordinate follicles > or =3mm, were classified as denuded, degenerated or healthy, and underwent IVM-IVF-IVC. The proportion of oocytes that developed to the blastocyst stage was higher (P<0.05) in those collected on Day 5 after wave emergence (23%) than on Day 2 (12%), 3 (13%) or 7 (16%). Data did not support the hypothesis of a local effect of the CL or dominant follicle. We conclude that a positive relationship exists between early follicular regression and oocyte competence. Moreover, morphologic characteristics of oocyte quality used in this study were not predictive in identifying competent oocytes.     

Effect of follicular fluid collected from various diameter follicles on the progression of nuclear maturation and developmental competence of pig oocytes

Supplementing in vitro maturation medium with porcine follicular fluid (FF) improves maturation rate, male pronucleus formation, and monospermic fertilization of pig oocytes. This study examined, (1) if there are differences in FF derived from large follicles (LF, 5–6 mm in diameter) and small follicles (SF, 3–4 mm in diameter) on the effect of supplementing the maturation medium with FF on the progression of nuclear maturation, fertilization rate, and developmental competence of porcine oocytes; (2) whether the FF source influences the effect of the FF on the maturation medium on the survival rate and proliferation rate of cumulus cells (CCs) and the expansion of cumulus-oocyte-complexes (COCs); (3) whether the oocyte source (oocytes collected from LFs or SFs) influences the effect of FF on the progression of the nuclear maturation of oocytes; (4) whether the factors in the FF that affect the kinetics of nuclear maturation are proteins, and the range of the molecular weight of the FF factors.

In experiment 1, adding FF from LFs (LFF) significantly accelerated nuclear maturation and improved the fertilization rate; the developmental ratio was comparable with those of adding FF from SFs (SFF). In experiment 2, adding LFF, but not SFF, improved the CC survival rate, although the FF source did not affect the proliferation rate. Expansion of COCs was greater with SFF than LFF. In experiment 3, LFF promoted nuclear maturation of oocytes collected from only LFs. There was a significant interaction between the FF source and the oocyte source in the effect on nuclear maturation stages at 36 h of maturation. In experiment 4, treatment of FF with heat or trypsin diminished the difference between the effect of LFF and SFF on the progression of nuclear maturation. In addition, the predominant effect of LFF compared to that of SFF on nuclear maturation was not affected by ultrafiltration of the FF with a 30-kDa filter, but was diminished by ultrafiltration with a 100-kDa filter. The present study suggests that some proteins present in LFF that range in molecular weight from 30 to 100 kDa improve the developmental competence of oocytes probably via progression of nuclear maturation and cumulus cells viability.