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 cryodevice type and donors' sexual maturity on vitrification of minke whale (Balaenoptera bonaerensis) oocytes at germinal vesicle stage

Germinal-vesicle-stage oocytes enclosed with compact cumulus cell layers (COCs) were recovered from adult or prepubertal minke whale ovaries, and were vitrified in a solution containing 15% ethylene glycol, 15% DMSO and 0.5 M sucrose using either a Cryotop or an open-pulled straw (OPS) as the cryodevice. The post-warm COCs with normal morphology were cultured for 40 h in a 390 mosmol in vitro maturation medium, and oocytes extruding the first polar body were considered to be matured. The proportion of morphologically normal COCs after vitrification and warming was higher when the COCs were cryopreserved by Cryotop (adult origin, 88.4%; prepubertal origin, 80.8%) compared with the OPS (adult origin, 67.7%; prepubertal origin, 64.2%). The oocyte maturation rate was higher in the adult/Cryotop group (29.1%) compared with those of the prepubertal/Cryotop group (14.4%), the adult/OPS group (14.3%) and the prepubertal/OPS group (10.6%). These results indicate that the Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales.     

Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability

The nuclear stage at which oocytes are cryopreserved influences further development ability and cryopreservation affects ultrastructure of both cumulus cells and the oocyte. In this work, we analyze the effects of vitrification at different nuclear and cytoplasmic maturation stages on the oocyte ultrastructure and developmental ability. Culture in TCM199 + PVA with roscovitine 25 M during 24h led to meiotic arrest (MA) in cumulus-oocyte complexes (COCs), while permissive in vitro maturation (IVM) was performed in TCM199, 10% FCS, FSH-LH and 17beta-estradiol for 24 h. Oocytes were vitrified using the open pulled straw method (OPS) with minor modifications. Fresh and vitrified/warmed COCs were fixed as immature, after IVM, after meiotic arrest (MA) and after MA + IVM. Vitrification combined with MA followed by IVM produced the highest rates of degeneration, regardless of the vitrification time. As a consequence, lower proportions of embryos cleaved in these groups, although differences were eliminated at the five-eight cell stage. Development rates up to day 8 were similar in all experimental groups, being significantly lower than those in fresh controls. Only oocytes vitrified after IVM were able to give blastociysts. The morphological alterations observed can be responsible for compromised development. More research is needed to explain the low survival rates of the bovine oocyte after vitrification and warming.     

Effect of different vitrification solutions and cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle (GV) stage

The cryopreservation of immature oocytes would generate a readily available, non-seasonal source of female gametes for research and reproduction. In domestic animals, the most promising results on oocyte cryopreservation have been reported in cattle, few studies have been conducted on buffalo. The aim of the present study was to compare the use of different vitrification solutions and various cryodevices on viability and developmental competence of buffalo oocytes vitrified at the germinal vesicle (GV) stage. Cumulus oocyte-complexes (COCs) obtained at slaughterhouse from mature buffalo ovaries were randomly divided into three main groups and vitrified by using either straw or open pulled-straw (OPS) or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol (GLY); VS1 or 20% EG + 20% dimethylsulfoxide (DMSO); VS2, respectively. Following vitrification and warming, viable COCs were matured in vitro for 22 h. Some COCs were denuded and stained with 1.0% aceto-orcein to evaluate nuclear maturation, whereas the others were fertilized and cultured in vitro for 7 days to determine the developmental competence. Although the recovery rate (64.9%) was the lowest in the oocytes vitrified by SSV using 20% EG + 20% DMSO as compared to the other groups, the best survival rate of the COCs was achieved in the same treatment (96.7%), which was significantly higher (P < 0.05) than those vitrified using traditional straws (71.8% in VS1 and 73.6% in VS2) or those vitrified using OPS and VS1 (73.9%). Furthermore, in the nuclear maturation test, the highest maturation rate (75.5%) was achieved in SSV vitrified COCs using 20% EG + 20% DMSO (VS2), which was similar to the controls (77.1%). Post IVF and embryo culture, the highest cleavage and blastocyst development rates were obtained in COCs vitrified in 20% EG + 20% DMSO using SSV (47.1% and 24.0%, respectively), which showed no difference from the controls (61.2% and 46.9%, respectively). Our results clearly show that the combination of SSV and 20% EG + 20% DMSO could be used effectively to vitrify GV stage buffalo COCs.     

Resveratrol treatment increases mitochondrial biogenesis and improves viability of porcine germinal-vesicle stage vitrified-warmed oocytes

Vitrification induces mitochondrial dysfunction in warmed oocytes, and degeneration and biogenesis of mitochondria are crucial for maintaining oocyte quality. The present study addresses a hypothesis that treatment of vitrified-warmed oocytes with resveratrol could improve the viability of oocytes by activating mitochondrial biosynthesis. Cumulus oocyte complexes (COCs) collected from gilt ovaries were vitrified, warmed, and cultured in a medium containing vehicle or 1 μM resveratrol. Resveratrol treatment improved survival, maturation, and mitochondrial membrane potential of vitrified-warmed oocytes, but did not improve the development into blastocysts after parthenogenetic activation. Resveratrol treatment increased mitochondrial synthesis, as determined by the expression levels of TOMM20 and mitochondrial DNA copy number, in vitrified-warmed oocytes, but not in non-vitrified oocytes. In addition, the effect of resveratrol treatment on mitochondrial synthesis was reduced by EX527, a SIRT1 inhibitor. Resveratrol treatment of vitrified-warmed oocytes increased the expression levels of genes involved in mitochondrial synthesis (TFAM, POLG, and PGC1α) and increased nuclear translocation of NRF2. Furthermore, vitrification induced mitophagy, as confirmed by a reduction in TOMM20 expression and decreased p62 aggregation, whereas resveratrol treatment did not affect these mitophagic features. In conclusion, vitrification induced mitochondrial clearance in oocytes, whereas activation of SIRT1 by resveratrol treatment facilitated the recovery of vitrified-warmed oocytes through the activation of mitochondrial synthesis.     

Ultrastructural changes of sheep cumulus-oocyte complexes following different methods of vitrification

To determine the ultrastructural changes of sheep cumulus-oocyte complexes (COCs) following different methods of vitrification, good quality isolated COCs (GV stage) were randomly divided into the non-vitrified control, conventional straw, cryotop and solid surface vitrification groups. In both conventional and cryotop methods, vitrified COCs were respectively loaded by conventional straws and cryotops, and then plunged directly into liquid nitrogen (LN2); whereas in the solid surface group, vitrified COCs were first loaded by cryotops and then cooled before plunging into LN2. Post-warming survivability and ultrastructural changes of healthy COCs in the cryotop group especially in comparison with the conventional group revealed better viability rate and good preservation of the ooplasm organization. However in all vitrification groups except the cryotop group, mitochondria were clumped. Solely in the conventional straw group, the mitochondria showed different densities and were extremely distended. Moreover in the latter group, plenty of large irregular connected vesicles in the ooplasm were observed and in some parts their membrane ruptured. Also, in the conventional and solid surface vitrification groups, cumulus cells projections became retracted from the zona pellucida in some parts. In conclusion, the cryotop vitrification method as compared with other methods seems to have a good post-warming survivability and shows less deleterious effects on the ultrastructure of healthy vitrified-warmed sheep COCs.     

Expression of maturation genes and their receptors during in vitro maturation of sheep COCs in the presence and absence of somatic cells of cumulus origin

The purpose of this study was to investigate the effects of somatic cells of cumulus origin (sCC) on gene expression and maturation of cumulus oocyte complexes (COCs) in vitro. Good quality (i.e., healthy-looking) isolated sheep COCs were randomly divided into two treatment groups: control (COC with no sCC) and coculture (COC with sCC). Nuclear maturation statuses of oocytes were assessed after 27 hours of in vitro culture. Moreover, the expression levels of growth differentiation factor 9 (GDF9), bone morphogenetic protein (BMP)15, BMP6, bone morphogenetic protein receptor II (BMPRII), activin like kinase 5 (ALK5) (transforming growth factor β receptor 1: TGFβR1), ALK6 (BMPR1b), activin A receptor, type IIB (ActRIIB), and ALK3 (BMPR1a), as well as hyaluronan synthase 2 (HAS2) and prostaglandin endoperoxide synthase 2 (Ptgs2) in the COCs were assessed in both treatment groups after 3 h and 27 h of culture. The results showed that the proportion of metaphase II (MII) stage oocytes was significantly higher in the coculture group compared with the controls (77.21% ± 1.17 vs. 67.49% ± 1.80; P < 0.05). The relative expressions of BMPRII, ALK6, and ActRIIB in control group and GDF9 and ActRIIB in coculture group showed significant differences during culture as assessed by real time polymerase chain reaction (P < 0.05). The mean expression levels of BMPRII, ALK5, ALK6, and ActRIIB mRNA were decreased in the coculture group compared with those in the control group after 27 h of culture (P < 0.05). In conclusion, we propose that in vitro maturation of sheep COCs alone disrupted the normal gene expression levels of both TGFβ ligands and receptors, and also reduced the maturation rate. Coculture with sCC enhanced the maturation rate of oocytes concomitantly with reduced gene expression levels of a number of TGFβ ligands and receptors.     

Factors affecting nuclear maturation, cleavage and embryo development of vitrified bovine cumulus-oocyte complexes

The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable.     

Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine

The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P < 0.05) than that of the liquid nitrogen group (81.1%). When the vitrified–thawed oocytes were matured in vitro for 24 h, the maturation rate in liquid helium group (50.6%) was higher (P < 0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P < 0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified–thawed oocytes were matured 24 h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes.     

Cryopreservation of Cattle Oocytes: Effects of Meiotic Stage, Cycloheximide Treatment, and Vitrification Procedure

Different parameters likely to influence the survival of bovine oocytes after a vitrification procedure were evaluated: oocyte meiotic stage, cycloheximide treatment at the beginning or the end of maturation, and three vitrification procedures using conventional straws, open pulled straws (OPS), or microdrops. For each procedure a mixture of cryoprotectants (25% ethylene glycol and 25% glycerol) was used. After the oocytes were warmed and subjected to in vitro maturation and fertilization, the number that developed into blastocysts was determined. Results show that cryoprotectant exposure reduced embryo development and that cycloheximide treatment had no beneficial effect on oocytes vitrified in conventional straws. Among the three vitrification procedures, only the OPS method yielded blastocysts (approximately 3% of vitrified oocytes) irrespective of their initial meiotic stage. This result highlights the major influence of the cooling rate in an oocyte vitrification protocol.     

Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

Patterns of expression of messenger RNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development and characterization of ovarian follicular populations in ewes carrying the Woodlands FecX2W mutation

Woodlands sheep have a putative genetic mutation (FecX2(W)) that increases ovulation rate. At present, the identity of FecX2(W) is unknown. The trait does not appear to be due to the previously described mutations in bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), or bone morphogenetic protein receptor type 1B (BMPR1B) that affect ovulation rate in sheep. Potentially, FecX2(W) could be an unidentified genetic mutation in BMP15 or in the closely related GDF9, which interacts with BMP15 to control ovarian function. Alternatively, FecX2(W) may affect ovulation rate by changing the expression patterns in the molecular pathways activated by genes known to regulate ovulation rate. The objectives of these experiments were to sequence the complete coding region of the BMP15 and GDF9 genes, determine the patterns of expression of mRNAs encoding GDF9, BMP15, TGFBR1, BMPR1B, and BMPR2 during follicular development, and characterize the follicular populations in ewes heterozygous for the Woodlands mutation and their wild-type contemporaries. No differences in the coding sequences of BMP15 or GDF9 genes were identified that were associated with enhanced ovulation rate. The expression patterns of GDF9 and BMPR2 mRNAs were not different between genotypes. However, expression of BMP15 mRNA was less in oocytes of FecX2(W) ewes in large preantral and antral follicles. Expression of ALK5 mRNA was significantly higher in the oocytes of FecX2(W) ewes, whereas expression of BMPR1B was decreased in both oocytes and granulosa cells of FecX2(W) ewes. FecX2(W) ewes also had increased numbers of antral follicles <1 mm in diameter. These follicles were smaller in average diameter, with the oocytes also being of a smaller mean diameter. Given that a mutation in BMP15 or BMPR1B results in increased ovulation rates in sheep, the differences in expression levels of BMP15 and BMPR1B may play a role in the increase in ovulation rate observed in Woodlands ewes with the FecX2(W) mutation.     

Oocyte-secreted factors enhance oocyte developmental competence

The capacity of fully grown oocytes to regulate their own microenvironment by paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) may in turn contribute to oocyte developmental competence. Here, we investigated if OSFs have a direct influence on oocyte developmental competence during in vitro maturation (IVM). Bovine cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured in serum-free medium. COCs were either co-cultured with denuded oocytes (DOs) or treated with specific OSFs: recombinant bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9). Following maturation, embryos were fertilized and cultured in vitro and blastocyst development and cell number were assessed on day 8. Co-culturing intact COCs with DOs did not affect cleavage rate, but increased (P<0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination from 39% to 51%. OSFs also altered blastocyst cell allocation as co-culture of COCs with DOs significantly increased total and trophectoderm cell numbers, compared to control COCs. BMP15 alone, GDF9 alone or the two combined all (P<0.05) increased the proportion of oocytes that reached the blastocyst stage post-insemination from 41% (controls) to 58%, 50% and 55%, respectively. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP15 and GDF9, respectively, neutralized the stimulatory effects of the exogenous growth factors and impaired the developmental competence of control COCs. These results demonstrate that OSFs, and particularly BMP15 and GDF9, enhance oocyte developmental competence and provide evidence that OSF regulation of the COC microenvironment is an important determinant of oocyte developmental programming.     

The proregion of mouse BMP15 regulates the cooperative interactions of BMP15 and GDF9

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are secreted by the mammalian oocyte and are essential for ovarian follicular development, ovulation, and fertility. However, the secreted forms of the BMP15 and GDF9 proteins and the nature of cooperative molecular interactions between BMP15 and GDF9 previously reported have not been fully characterized. In this study, we found that recombinant mouse BMP15 and GDF9 are secreted as cleaved mature and proregion proteins, with BMP15 also secreted as uncleaved promature protein. Noncovalent interactions were identified between the mature and proregion proteins of each growth factor. Moreover, GDF9 mature protein was found to coimmunoprecipitate with the BMP15 proregion, suggestive of a heteromeric association between BMP15 and GDF9. Mouse GDF9 was found to exist mostly as a dimer of mature protein, in both the presence and absence of BMP15. In contrast, BMP15 formed mostly multimers of proregion and mature protein when combined with GDF9, providing further evidence for heteromeric interaction. Mouse BMP15 was found to act cooperatively with GDF9 in a rat granulosa cell thymidine incorporation bioassay and to signal through the BMPR2 and ACVR1B/TGFBR1/ACVR1C receptor-mediated pathways. Immunoneutralization experiments using GDF9 mature protein antibody indicated that these cooperative interactions are species specific. Additionally, immunoneutralization with proregion antibodies highlighted the involvement of the BMP15 proregion in BMP15/GDF9 cooperative interactions. Taken together, these findings support a novel hypothesis where the extracellular cooperative interactions of recombinant mouse BMP15 and GDF9 are multimeric, involving the proregion of BMP15, and may well be species specific.     

Vitrification devices affect structural and molecular status of in vitro matured ovine oocytes

We evaluated the effect of three different cryodevices on membrane integrity, tubulin polymerization, maturation promoting factor (MPF) activity and developmental competence of in vitro matured (IVM) ovine oocytes. IVM oocytes were exposed during 3 min to 7.5% DMSO and 7.5% ethylene glycol (EG) in TCM199 and 25 sec to 0.5 M sucrose, 16.5% DMSO and 16.5% EG, loaded in open pulled straws (OPS), cryoloops (CL) or cryotops (CT) and immersed into liquid nitrogen. Untreated (CTR) or exposed to vitrification solutions but not cryopreserved (EXP) oocytes were used as controls. After warming, double fluorescent staining evidenced a lower membrane integrity in vitrified groups compared to the controls (P < 0.01). After in vitro fertilization and culture OPS and CL groups evidenced a lower cleavage rate than CT and controls (P < 0.01) while blastocysts were obtained only in CL and EXP, at a lower rate than CTR (P < 0.01). All vitrified groups showed alterations in spindle conformation, which were partially recovered in OPS and CT groups. MPF activity was lower in treated compared to CTR and CT showed the lowest value (P < 0.01). After 2 hr culture MPF activity was restored in all groups except CT. Parthenogenetic activation was higher in treated compared to CTR and CT evidenced the highest value. Our results indicate that cryodevice influences not only the ability to survive cryopreservation but is also associated with molecular alterations which affect developmental competence.     

Ultrastructure of bovine oocytes exposed to Taxol prior to OPS vitrification

Our objective was to document potential subcellular consequences of treatment with the microtubule stabilizer Taxol with or without subsequent vitrification of cow and calf oocytes by the open pulled straw (OPS) method. Oocytes were divided into four experimental groups for cows and four groups for calves: (1) a control group fixed immediately after maturation; (2) an OPS group cryopreserved by conventional OPS; (3) a Taxol/CPA group exposed to 1 microM Taxol and cryoprotective agents (CPAs); and (4) a Taxol/OPS group vitrified by OPS including 1 microM Taxol to the vitrification solution. All oocytes were processed for light and transmission electron microscopy. The main injuries were observed on the metaphase plate and the spindle. In control oocytes, the metaphase appeared as condensed chromosomes arranged in a well-organized metaphase plate and the spindle showed well organized microtubules in both cow and calf oocytes. However, in cow OPS oocytes, the metaphase plate was disorganized into scattered chromosomes or the chromosomes were condensed into a single block of chromatin. In addition, microtubules were not organized as typical spindles. In contrast, cow Taxol/OPS oocytes as well as both cow and calf Taxol/CPAs oocytes showed well-organized metaphase plates and normal spindle morphology. All calf OPS and calf Taxol/OPS oocytes displayed a single block of chromatin and no microtubules could be observed around the chromosomes. In conclusion, treatment with 1 microM Taxol before and during vitrification did not induce adverse changes in the oocyte cytoplasm or metaphase spindles in adult bovine oocytes, but stabilized the metaphase and spindle morphology.     

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.