Oocyte secreted factors improve embryo developmental competence of COCs from small follicles in prepubertal goats

Oocytes secrete soluble paracrine factors called Oocyte Secreted Factors (OSFs) which regulate the cumulus cell phenotype. Follicle populations in ovaries from prepubertal females have smaller diameters than their adult counterparts. Oocytes from small follicles are less competent than those from large follicles. The aim of this study was to investigate, in prepubertal goats, the effect of OSFs secreted by denuded oocytes (DOs) from small (<3 mm) or large (≥3 mm) follicles during IVM on embryo development and the blastocyst quality of cumulus-oocyte complexes (COCs) from small follicles and to determine if GDF9 participates in this process. Treatment groups were: (A) COCs non selected by their follicle size (control group); (B) cumulus oocytes complexes from small follicles (SFCOCs), (C) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from small follicles (SFCOCs + SFDOs), and (D) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from large follicles (SFCOCs + LFDOs). The effect of the addition of kinase inhibitor SB-431542, which antagonizes GDF9, was tested in A, C, and D treatment groups. Co-cultured SFCOCs with SFDOs or LFDOs significantly augmented the blastocyst rate in comparison to SFCOCs alone (15.77%, 17.39% vs. 10.31%, respectively). Blastocysts from SFCOCs + LFDOs group showed higher rates of tetraploid nuclei than blastocysts from SFCOCs and the control group (14.43% vs. 5.45% and 5.24%, respectively; P < 0.05). However, we did not observe differences in the hatching rate, mean cell number or embryo cryotolerance (P > 0.05) between the four treatment groups. The addition of SB-431542 during IVM did not have any effect on blastocyst rate (P > 0.05). In conclusion, in prepubertal goats, COCs with a low embryo developmental competence as a consequence of follicle size can be improved by coculturing them with denuded oocytes from both small and large follicles. GDF9 does not seem play a role in this improvement.     

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.     

Effect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes

The aim of the study was to evaluate meiotic maturation, and expression of genes coding for oocyte secreted factors (GDF9, BMP15, TGFBR1, and BPR2) and apoptosis (BCL2, BAX and P53) after vitrification of immature goat cumulus oocyte complexes (COCs) and in vitro maturation. COCs were vitrified in a solution containing ethylene glycol, dimethyl sulfoxide and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). Freshly collected COCs (Control), COCs exposed to vitrification and dilution solutions without cryopreservation (EC) and vitrified-warmed COCs were matured in vitro for 27h. The viability of vitrified-warmed COCs 2 h post warming and in vitro maturation was similar for CL, HS and CT. The proportion of oocytes that extruded a 1st polar body and reached TI/MII was significantly higher with CT and HS followed by CL, OPS and CS. Gene expression of GDF9, BMP15, BMPR2, BAX and P53 were comparable to control levels for OPS, CL, HS and CT. The gene expression pattern in CS vitrified COCs was by contrast changed in that GDF9, BMP15, TGFBR1 and BAX were up regulated and BMPR2, BCL2 and P53 down regulated. In conclusion immature goat COCs vitrified using CT and HS showed that viability, maturation rates and expression of genes coding for oocyte secreted factors and apoptosis were similar to non-vitrified controls.     

Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.     

Porcine cumulus cell influences ooplasmic mitochondria-lipid distributions, GSH-ATP contents and calcium release pattern after electro-activation

The objective was to explore mechanisms of the influence of porcine cumulus cells (CC) on oocyte maturation. Immature porcine oocytes were matured in groups of denuded oocyte (DOs), cumulus-oocyte complexes (COCs), denuded oocytes co-cultured with CC (DoCC), or with cumulus-oocyte complexes (DoCOCs). Ooplasmic mitochondria-lipid distributions, glutathione (GSH)-adenosine triphosphate (ATP) contents, calcium release pattern, and developmental competence after parthenogenetic activation were assessed after IVM. The portion of matured oocytes after IVM and the developmental competence and GSH content in single oocytes were lower in DOs than in COCs (P < 0.05). In contrast, the maturation rate and development in DoCOCs and COCs were higher than in DoCC and DOs (P < 0.05). The blastocyst rate in DoCOCs was higher than in DOs (P < 0.05), and ATP content in COCs was higher than in all other groups (P < 0.01). In addition, the rate of oocytes with damaged oolemma in DOs (35%) was significantly higher than in COCs (3%), DoCOCs (7%), and DoCC (10%). The rate of oocytes with evenly distributed mitochondria was 70% in DOs, which was significantly lower than in COCs and DoCC (89 and 84%, respectively). The percentage of oocytes with normal lipid droplets distributions in COCs (70%) was significantly higher than in three other groups, whereas both percentages in DoCC and DoCOCs were higher than in DOs (P < 0.05). The duration of [Ca²⁺] rise in DOs was longer than in three other groups, whereas the duration was shortest in COCs. The amplitude of the [Ca²⁺] rise in DOs was significantly lower than in other groups (P < 0.05), but the amplitude did not differ significantly among DoCC, DoCOCs and COCs. In conclusion, the presence of porcine CC during IVM functionally affected ooplasmic mitochondria-lipid distributions and GSH-ATP contents, which may affect the calcium release pattern and developmental competence of oocytes after electro-activation.     

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.     

Effects of Upregulation of Hsp27 Expression on Oocyte Development and Maturation Derived from Polycystic Ovary Syndrome

Heat shock protein 27 (Hsp27) is a heat shock protein family member which can inhibit apoptosis. Our previous studies reported down-regulated Hsp27 in ovarian tissue derived from women with polycystic ovary syndrome (PCOS) however, the exact effect of Hsp27 on oocyte maturation and developmental competence in PCOS is unclear. The effect of Hsp27 over-expression was studied in vitro using oocytes derived from PCOS patients. An artificial GFP-plasmid was injected into human oocyte to increase Hsp27 protein level. Oocyte maturation was evaluated by morphological observation. Mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and embryonic developmental competence was evaluated. Critical apoptotic factors and cytokines were measured at both the mRNA and protein level. Our results revealed that Overexpression of HSP27 lowered the maturation rate of oocytes derived from PCOS patients. Meanwhile, fertilization rate and high quality embryo rate were similar between the Hsp27 overexpressing group and controls; however, the blastocyst formation rate in this group was significantly higher than control. Expression analysis revealed that the oocyte-secreted factors, BMP15 and GDF9, and the apoptotic-related regulators, Caspase 3, 8 and 9, were all significantly decreased in Hsp27 overexpressing oocytes. In conclusion, upregulation of Hsp27 inhibits oocyte maturation from PCOS patients, but improves embryonic developmental potential.     

Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats

The aim of the study was to determine whether the selection of immature oocytes by a combination of cumulus-oocyte-complexes (COCs) morphology and staining with brilliant cresyl blue (BCB) would be helpful in selecting developmentally competent oocytes, and thereby increase the efficiency of blastocyst production from ovarian oocytes of FSH-primed, adult goats. In a second experiment the interaction between oocyte quality and semen donor was assessed. In a third experiment the usefulness of Vero cells for co-culture with goat embryos was investigated. In the pool of morphologically normal COCs recovered from ovaries following slicing (21.9+/-11.0), the mean rate of COCs classified as BCB+ was 85.6%, and the BCB- was approximately 11%. Oocytes classified as grade 1 and BCB+ exhibited the highest developmental competence (P<0.001) after in vitro maturation and fertilization compared with oocytes of grade 1 BCB- and grade 2 BCB+ or BCB-. There were no significant differences in developmental competence in grade 2 oocytes, regardless of BCB coloration. No significant differences in embryo cleavage and blastocyst formation rates among three bucks were observed when morphologically normal, BCB+ oocytes were used. For all tested bucks, differences in embryo production efficiency were related only to the oocyte quality. Similar blastocyst rates were developed from embryos co-cultured with goat oviduct epithelial cells (34.3%) and with Vero cells (33.3%). These results show that the most important criterion for selection of COCs before maturation is the visual assessment of morphological features. Staining with BCB of COCs recovered from adult goats does not enhance efficiency of selection of developmentally competent oocytes for IVF.     

Coculturing denuded oocytes during the in vitro maturation of bovine cumulus oocyte complexes exerts a synergistic effect on embryo development

The present study examined the effect of coculturing cumulus oocyte complexes (COCs) and denuded oocytes (DOs) during in vitro maturation (IVM) on nuclear and cytoplasmic maturation, zona pellucida (ZP) hardening, the pattern of fertilization and glutathione peroxidase 1 (GPX1) gene expression in the oocyte. Furthermore, the rate of embryonic development and the quality of blastocysts were examined for both COCs and DOs. Three IVM conditions were studied: 1) the coculture of 12 COCs and 60 DOs, 2) COC control with 12 COCs, and 3) DO control with 60 DOs. The IVM was performed in a 120-μl droplet of TCM199-based IVM medium. Following IVM, in vitro fertilization (IVF) and in vitro culture (IVC) were conducted separately for the COCs and DOs (DO coculture) from the IVM coculture group. Coculturing COCs and DOs increased the percentage of oocytes reaching the blastocyst stage and the total number of cells per blastocyst in both the COC coculture (44.4 ± 8.6 vs 26.7 ± 9.7%, P < 0.01, and 137.9 ± 24.9 vs 121.7 ± 21.1, P < 0.05) and the DO coculture (20.5 ± 5.0 vs 11.1 ± 2.5%, P < 0.01, and 121.9 ± 27.5 vs 112.3 ± 33.2, P < 0.05) compared to their respective control groups. The synergistic effects of coculturing were detected as increased nuclear and cytoplasmic maturation, the prevention of ZP hardening, increased monospermic fertilization and increased expression of GPX1 in the oocytes in response to endogenous oocyte-secreted factors. In conclusion, coculturing COCs and DOs may be an effective culture system for both intact COCs and immature DOs.     

Differential gene expression of bone morphogenetic protein 15 and growth differentiation factor 9 during in vitro maturation of porcine oocytes and early embryos

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) belong to the TGF-beta superfamily and are involved in the regulation of folliculogenesis. Though there are many reports concerning the expression and regulation of GDF9 in the process of oocyte maturation, expression of BMP15 during oocyte maturation is still not clearly understood. It has been reported that BMP15 and GDF9 expression is important in folliculogeneiss and that the regulation of these two proteins is complex and species-specific. In this report, we investigated the expression of BMP15 and GDF9 genes during in vitro maturation (IVM) at 0, 6, 12, 18, 24, 30, 36, 42 and 48 h for porcine oocytes. Porcine GDF9 gene was found to be highly expressed in immature oocytes and declined slowly during the oocyte maturation process. BMP15mRNA and its encoded protein were expressed at low levels in immature oocytes and increased to the highest level at 18 h of IVM, which coincides with the time of cumulus cell expansion. Thus, these two genes were differentially expressed during the oocyte maturation process and BMP15 is specifically expressed during cumulus cell expansion in porcine oocytes.     

Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells

The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the cooling-and-freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO) cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs). The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization.     

Prepubertal bovine oocyte: A negative model for studying oocyte developmental competence

To identify potential markers of maturation quality, differences in developmental capacity between cow and calf oocytes were compared in parallel with their constitutive and neosynthetic protein profiles before and after in vitro maturation (IVM). A comparison was also made between the protein profiles of follicular fluid (FF) from calf and cow ovaries. The effect of epidermal growth factor (EGF) during IVM on the subsequent development of prepubertal calf oocytes was examined. The effect of the presence of fetal calf serum (FCS) during development of embryos originating from calf oocytes was also examined. No differences were noted between the constitutive proteins of cow and calf oocytes and only a minor modification was observed before IVM in the pattern of neosynthesized proteins (presence of a band of 37 kD and a slight increase in the intensity of band of 78 kD in cow as compared to calf oocytes). However, the comparison of constitutive protein profiles from calf and cow FF demonstrated quantitative (the bands of 34 and 45 kD were more intense for cow than for calf) differences. EGF receptors (EGF-R) were demonstrated on cumulus—oocytes complexes (COCs) by immunofluorescence. There was no difference in intensity between cow and calf COCs. Furthermore, the addition of EGF during IVM of calf oocytes dramatically stimulated cumulus expansion and significantly increased the cleavage rate at 72 h post-insemination (82% vs 67%), as well as the proportion of embryos at the 5- to 8-cell stage at this time (54% vs 43%). Also, blastocyst yields at day 6 (11% vs 5%) and at day 8 (17% vs 10%) were significantly higher in the presence of EGF P < 0.05). The addition of FCS to synthetic oviduct fluid droplets at day 2 of culture (48 hpi) had no effect on cleavage, blastocyst yield, or blastocyst cell number. In conclusion, differences in developmental ability between calf and cow oocytes would appear to be not solely linked to differences in oocyte protein patterns. It is likely that the FF, which constitutes the microenvironment in which the oocyte develops, plays a major modulating role in determining the fate of the oocyte/follicle. © 1996 Wiley-Liss, Inc.     

Supplementation of maturation medium with CoQ10 enhances developmental competence of ovine oocytes through improvement of mitochondrial function

In vitro maturation (IVM) can impair the balance between antioxidant capacity and oxidative stress, and jeopardize embryo development by increasing oxidative stress, reducing energy metabolism, and causing improper meiotic segregation. Balancing the energy production and reduction of oxidative stress can be achieved by supplementation with coenzyme Q10 (CoQ10), an electron transporter in the mitochondrial inner membrane. To improve the in vitro production of ovine embryos, we studied the effect of CoQ10 supplementation during the maturation of sheep oocytes. A minimum of 100 cumulus‐oocyte complexes (COCs) were matured in the presence of 15, 30, or 50 μM CoQ10 in three to five replicates; next, in vitro fertilization and culture in a subset of oocytes were done. Our data revealed that compared to control oocytes or other concentrations of CoQ10, supplementation with 30 µM CoQ10 resulted in a significant increase in blastocyst formation and hatching rates, improved the distribution, relative mass and potential membrane of mitochondria, decreased the levels of reactive oxygen species and glutathione and lessened the percentage of oocytes with misaligned chromosomes after spindle assembly. The relative expression levels of apoptosis markers CASPASE3 and BAX were significantly reduced in CoQ10‐treated oocytes and cumulus cells whereas the relative expression level of GDF9, an oocyte‐specific growth factor, significantly increased. In conclusion, supplementation with CoQ10 improves the quality of COCs and the subsequent developmental competence of the embryo.     

Oocyte maturation: emerging concepts and technologies to improve developmental potential in vitro

Oocyte in vitro maturation (IVM) is an important reproductive technology that generates mature oocytes that are capable of supporting preimplantation embryo development and full development to term. There is great clinical and commercial incentive to improve the efficiency of the technology, however, progress has been slow over the past decade. A critical challenge is to understand what constitutes oocyte developmental competence and the mechanisms governing it. We have taken the approach of studying in detail oocyte-somatic cell interactions; including, oocyte-cumulus cell (CC) gap-junctional communication, and bidirectional paracrine signalling between the two cell types. It is becoming clear that, compared to oocytes matured in vivo, IVM oocytes undergo maturation prematurely as they are still in the process of acquiring developmental competence in vivo, and the molecular cascade reinitiating meiosis differs entirely to that in vivo. Attempts to enhance oocyte developmental competence by attenuating the spontaneous meiotic resumption of oocytes in vitro have been met with mixed success. Kinase inhibitors that prevent maturation-promoting factor activity have, in general, been ineffectual on promoting oocyte developmental potential post-IVM. In contrast, agents that modulate oocyte cAMP during IVM show greater potential, possibly as these compounds extend oocyte-CC gap-junctional communication. An important concept that is now emerging is that the oocyte secretes potent growth factors that regulate fundamental aspects of CC function and thereby determine the distinctive phenotype of the cumulus-oocyte complex. The capacity of an oocyte to regulate its own microenvironment by oocyte-secreted factors (OSFs) may constitute an important component of oocyte developmental competence. In support of this notion, we have recently demonstrated that supplementing IVM media with exogenous OSFs improves oocyte developmental potential, as evidenced by enhanced pre- and post-implantation embryo development.     

Development of normal mice after microinjection of round spermatids into oocytes stored at room temperature for one day

Early studies have shown that some mouse cumulus-oocyte complexes (COCs) stored at room temperature for 24 hr still retained full developmental potential. In this study, we stored mouse COCs and denuded oocytes (DOs) at room temperature for 24 hr and activated these oocytes with 10 mM SrCl(2) or injected the oocytes with round spermatids. We found that DOs were better than COCs when stored at room temperature for 1 day and more normal oocytes were obtained when COCs were stored in more H-CZB medium at room temperature for 1 day. The rates of normal oocytes were significantly different after preservation with three schemes (90.01%, 55.81%, and 86.70%, P < 0.05). Our results also indicated that oocytes stored at room temperature for 1 day were fertilized normally (extrusion of the second polar body and formation of male and female pronuclei [PN]) after microinjection of round spermatid nuclei, and that the existence of cumulus cells (CCs) during oocyte storage did not significantly influence the early cleavage but had a detrimental effect on later embryo development and full-term development. After fertilization, most embryos developed to two-cell stage after being cultured for 24 hr, and the development rates of four- to eight-cell embryos between two experiments were similar. However, the rates of morula/blastocyst formation were significantly different (47.44% and 26.27%, respectively, P < 0.05). The birth of four healthy pups from stored DOs indicated that the storage of DOs at room temperature for 1 day might become a practical procedure in mammalian reproduction.     

Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes

The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.     

Developmental competence of porcine oocytes selected by brilliant cresyl blue and matured individually in a chemically defined culture medium

The objective of this study was to investigate the effects of oocyte selection using brilliant cresyl blue (BCB) and culture density during individual in vitro maturation (IVM) on porcine oocyte maturity and subsequent embryo development using a chemically defined medium. Cumulus-oocyte complexes (COCs) were classified as BCB-positive or BCB-negative after exposure to a BCB solution for 90 min. The classified COCs were matured in a group (15 COCs per 100-μL droplet) or individually (1 COC per 1-, 2.5-, 5-, or 10-μL droplet). Meiotic competence, intraoocyte glutathione concentration, and developmental competence after intracytoplasmic sperm injection were monitored. The BCB selected oocytes competent for nuclear and cytoplasmic maturation. Furthermore, meiotic competence for oocytes matured individually in a 5-μL droplet was superior (P < 0.05) to that of oocytes matured in a 1-μL droplet. Also, the culture density in a 5-μL droplet during IVM resulted in a higher (P < 0.05) rate of cleaved embryos than that in a 1-μL droplet and produced a similar rate of blastocysts compared with that of a group culture system. Conversely, BCB selection did not improve cleavage and blastocyst formation. In conclusion, it was possible to predict porcine oocytes competent for maturation using oocyte selection with BCB. Moreover, a 5-μL droplet during the individual IVM culture was most suitable for oocyte maturation and subsequent embryo development, although every culture density used in this study supported development up to the blastocyst stage.