Maturation Of Bovine Cumulus-oocyte Complexes Research Articles

Energy metabolism disorders during in vitro maturation of bovine cumulus-oocyte complexes interfere with blastocyst quality and metabolism

Glucose and fatty acids (FA) metabolism disturbances during oocyte in vitro maturation (IVM) affect their metabolism and surrounding cumulus cells, but only inhibition of glucose metabolism decreases embryo culture efficiency. Therefore, the present experiment aimed to reveal if glucose or FA metabolism inhibition leads to the disruption of embryo developmental potential, and to characterize the metabolic landscape of embryos reaching the blastocyst stage. Inhibitors of glucose (IO + DHEA) or FA (ETOMOXIR) metabolism were applied during IVM, and the control group was matured under standard conditions. Blastocysts obtained from experimental and control groups were analyzed with regard to lipidome and metabolome (mass spectrometry), transcriptome (RNA-Seq) and fluorescence lipid droplets staining (BODIPY). We showed that inhibition of glucose and fatty acid metabolism leads to cellular stress response compromising the quality of preimplantation embryos. The inhibition of energy metabolism affects membrane fluidity as well as downregulates fatty acids biosynthesis and gene expression of trophectoderm cell line markers. Therefore, we conclude that oocyte maturation environment exerts a substantial effect on preimplantation development programming at cellular and molecular levels.

Effects of cyclic adenosine monophosphate modulating agents during oocyte pre-maturation and the role of melatonin on in vitro maturation of bovine cumulus-oocyte complexes

This study investigated the effects of cyclic adenosine monophosphate modulating during cumulus-oocyte complexes (COCs) pre-maturation and the role of melatonin on in vitro maturation (IVM) of bovine COCs. In experiment one, COCs were pre-matured for 8 h in control medium or with 3-isobutyl-1-methylxanthine (IBMX) and forskolin, IBMX and C-type natriuretic peptide, c-type natriuretic peptide and forskolin or IBMX, forskolin and c-type natriuretic peptide. Then, meiotic progression was evaluated. In experiment two, COCs were pre-matured, followed by IVM in control medium alone or with 10−6, 10−7 or 10−8 M melatonin. After IVM, chromatin configuration, transzonal projections (TZPs), reactive oxygen species, mitochondrial distribution, ultrastructure and mRNA expression for antioxidant enzymes were evaluated. In experiment 1, COCs pre-matured with both C-type natriuretic peptide and forskolin or C-type natriuretic peptide, forskolin and IBMX had lower meiotic resumption rate when compared to control. Considering that IBMX had not an additional effect to potentiate inhibition of meiotic resumption, a combination of C-type natriuretic peptide and forskolin was chosen. In experiment 2, COCs matured with 10−8 M melatonin had greater rates of meiotic resumption when compared to the other treatments (P < 0.05). The COCs matured with 10−7 or 10−8 M melatonin had greater mitochondrial activity (P < 0.05), while those matured with 10−6 or 10−8 M of melatonin had greater levels of TZPs. Ultrastructure of oocyte and cumulus cells after IVM with melatonin was relatively well preserved. COCs matured with 10−8 M melatonin increased mRNA expression for superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), when compared to non-cultured and pre-matured COCs, respectively. In conclusion, bovine COC pre-maturation with C-type natriuretic peptide and forskolin, followed by IVM with 10−8 M melatonin improves meiotic resumption rates, TZPs, mitochondrial distribution and mRNA expression for SOD and CAT.

Effect of lycopene supplementation to bovine oocytes exposed to heat shock during in vitro maturation

We investigated the effect of the antioxidant lycopene supplemented into the in vitro maturation medium (TCM-199 with 20 ng/mL epidermal growth factor and 50 mg/mL gentamycin) in a heat shock (HS) model to mimic in vivo heat stress conditions. Bovine cumulus-oocyte complexes were supplemented with 0.2 μM lycopene (or not supplemented; control) under HS (40.5 °C) and non-HS (NHS; 38.5 °C) during maturation. After 22 h of maturation, we evaluated the nuclear status of the oocytes, the level of reactive oxygen species (ROS) production, and the respective blastocyst development and quality (via differential staining). Data were fitted in logistic and linear regression models, and the replicates were set as a random effect. The nuclear maturation was higher in NHS (84.0 ± 3.2%; least square mean ± standard error) than HS control (60.4 ± 4.3%; P < 0.001). Remarkably, the nuclear maturation in HS lycopene (71.7 ± 4.1%) was similar to NHS control (P = 0.7). Under HS conditions lycopene reduced ROS production (27.4 ± 4.8; relative fluorescence units (RFU)) in comparison to HS control (33.8 ± 1.8 RFU; P = 0.009). However, the ROS production in NHS lycopene (18.9 ± 2.0 RFU) was similar to NHS control (18.7 ± 1.8 RFU; P = 0.9). The cleavage rate in HS lycopene (76.1 ± 3.3%) was not lower than NHS lycopene (83.3 ± 2.5%; P > 0.1). On the day 8 of embryo development, the blastocyst rate was higher for NHS lycopene (55.2 ± 4.7%) versus NHS control (44.5 ± 4.7%; P = 0.04), but under HS the day 8 blastocyst rate was similar between control (29.9 ± 4.2%) and lycopene (32.3 ± 4.2%; P = 0.9). Lycopene supplementation increased the cell number of the embryos (total cell, trophectoderm, and inner cell mass numbers) under NHS conditions (P > 0.03). The apoptotic cell ratio was lower in lycopene (NHS and HS) versus control (NHS and HS) (P > 0.04). Lycopene has the ability to scavenge oocyte ROS and improved the cleavage rate of embryos under HS conditions. However, this could not be translated to a higher blastocyst development, which remained lower under HS. Results of our study indicate that antioxidant supplementation like lycopene during the maturation of bovine cumulus-oocyte complexes may be routinely used to improve blastocyst rate and quality under standard maturation conditions.

Corpora lutea affect in vitro maturation of bovine cumulus-oocyte complexes and embryonic development after fertilization with sex-sorted or conventional semen.

Influence of both the presence of a corpus luteum on the ovary and semen sex-sorting on development following in vitro fertilization is not yet conclusive. To determine the effect of these factors, 376 bovine oocytes were processed in vitro according to luteal presence on the ovary (CL+ and CL-) and type of semen used (sexed or conventional). Maturation rate was higher (P < 0.01) in CL- (136/138; 98.6%) than in CL+ (217/238; 91.2%). Cleavage rate was lower (P < 0.01) in CL+ with sexed semen (60/172; 34.9%) than in CL- with sexed semen (42/71; 59.1%), CL+ with conventional semen (47/66; 71.2%), and CL- with conventional semen (54/67; 85.1%). Compaction was similar (P = 0.69) in CL- (49/99; 49.4%) and CL+ (50/107; 46.7%). Blastulation rate was higher (P < 0.01) in CL- (26/99, 26.2%) than in CL+ (13/107; 12.1%) group. Expansion rate was higher (P = 0.01) in CL- (22/99; 22%) than in CL+ (11/107; 10.2%) group. Compaction rates were similar (P = 0.78) in sex-sorted (50/102; 49.0%) or conventional semen (49/104; 47.1%) groups. Blastulation was also similar (P = 0.91) with sex-sorted semen (19/102; 18.6%) and conventional semen (20/104; 19.2%). The rate of expanded blastocysts was similar (P = 0.89) in sex-sorted (16/102; 15.6%) and conventional (17/104; 16.3%) semen groups. In conclusion, the presence of CL can compromise maturation of the oocytes and their development, as a higher proportion of cleavage-stage embryos can be obtained with non-sexed semen with oocytes from ovaries without a CL.

47 Inhibition of endoplasmic reticulum stress during invitro maturation improves the developmental competence of bovine cumulus-oocyte complexes

Endoplasmic reticulum (ER) stress, a dysfunction in protein-folding capacity of ER, is involved in many physiological responses including embryonic development. Evidence shows that the ER stress-induced unfolded protein response signaling pathway is associated with impairment of oocyte maturation and pre-implantation embryonic development; supplementation of culture medium with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, improved the developmental process of oocytes and embryos by attenuating ER stress. However, no reports are available on the role of TUDCA in reducing ER stress during IVM of bovine oocytes. Therefore, the aim of this study was to examine the mechanism of TUDCA on reducing ER stress in maturation of bovine cumulus-oocyte complexes (COCs) and whether inhibition of ER stress during maturation can promote subsequent embryonic development. Bovine ovaries were collected from a local slaughterhouse, and after aspiration COCs were matured with/without TUDCA (50, 100, and 200 µM) for 22h at 38.5°C in a humidified atmosphere of 5% CO2. After IVM, we examined the maturation rate, reactive oxygen species, apoptosis, protein/mRNA expression levels, and subsequent embryonic development after IVF. The data were analysed using analysis of variance followed by the Tukey-Kramer multiple comparison test. As a result, the dose-dependent experiment shows that a 100μM concentration of TUDCA significantly increased the maturation rate and decreased the percentage of apoptotic cells in COCs and reactive oxygen species levels in denuded oocytes. Subsequently, the expression of ER stress inducible protein GRP78/BIP significantly decreased in COCs treated with 100 µM TUDCA compared with the control COCs. In addition, the mRNA expression of ER stress and pro-apoptotic markers (GRP78/BIP, PERK, IER1, ATF4, XBP1, CHOP, and BAX) in COCs were significantly decreased by TUDCA (100 µM) treatment, whereas it increased anti-apoptotic BCL2 expression. Moreover, we show that TUDCA (100 µM) supplementation enhances embryonic development by significantly increasing the blastocyst formation rate (43.6±1.8% vs. 49.7±1.3%) and decreasing the number of apoptotic cells (7.7±1.1% vs. 5.03±0.6%) in blastocysts. These findings suggest that existence of ER stress during maturation alters the developmental competence of bovine COCs. Therefore, for the first time, we demonstrate that application of TUDCA during IVM plays a crucial role in reducing ER stress and improves the meiotic maturation, oocyte quality, and subsequent embryonic development invitro.

164 Evaluation of the lipid content of in vitro-matured bovine cumulus - oocyte complexes in the presence of natriuretic peptides A, B, and C types

One of the difficulties still observed in in vitro production (IVP) of bovine embryos is the lower cryotolerance of such embryos, which has been related to their increased lipid accumulation during culture in the presence of fetal bovine serum (FBS). Previous studies have indicated that the cyclic guanosine monophosphate (cGMP) pathway may be involved in the lipid metabolism of bovine cumulus-oocyte complexes (COC). Synthesis of cGMP can be caused by activation of membrane guanylate cyclase (mGC), also called natriuretic peptide receptors (NPR1 and NPR2), which are activated by natriuretic peptides (NP) A, B, and C types (NPPA, NPPB, and NPPC). The objective of this study was to investigate the influence of supplementation with NP during in vitro maturation (IVM) on lipid content and nuclear maturation of bovine COC. Pools of 25 COC were submitted to IVM in TCM-199 with 0.2mM sodium pyruvate, 10μg mL−1 gentamicide, 0.5μg mL−1 FSH, 10% FBS, and NPPA (10−5 M), NPPB (10−7 M), or NPPC (10−5 M). The control group was matured without NP. After 24h, cumulus cells (CC) were removed and oocytes (OO) were fixed and permeabilized in 4% paraformaldehyde+0.5% Triton for 20min, stained with 10μg mL−1 Hoechst 33342 for 15min and 1μg mL−1 Nile Red for 30min. Then, the OO were placed in 13μL of ProLong (Thermo Fisher Scientific, Waltham, MA, USA) on glass slides, covered with a coverslip, and submitted to epifluorescence microscopy to evaluate nuclear maturation (emission 445-450nm and excitation 475-490nm) and lipid content (emission 590nm and excitation 516-560nm). Data for 4 replicates/group were tested for normality of results and homogeneity of variance, and then submitted to ANOVA, followed by Tukey test using a GraphPad Prism software (GraphPad Inc., San Diego, CA, USA), with a significance level of 5%. Nuclear maturation was influenced only by one of the NP added to IVM medium, NPPC, which reduced maturation rate (68.3% metaphase II, MII) compared with the control (82.7% MII; P&amp;lt;0.05). Maturation rates of NPPA (79.5% MII) and NPPB (85.1% MII) did not differ from the control (P&amp;gt;0.05). Activation of mGC by NPPB generated oocytes with lower lipid content (34.02±1.2 IF/μm2) compared with control (36.98±0.7 IF/μm2; P&amp;lt;0.05). Neither NPPA (36.5±1.4 IF/μm2) nor NPPC (39.3±1.4 IF/μm2) was able to reduce the lipid content in oocytes matured in vitro relative to control (P&amp;gt;0.05). Different NP may have different effects on maturation and lipid content in in vitro matured bovine oocytes; NPPB may be favourable for reducing the lipid content of matured bovine oocytes in vitro.

The effect of lysophosphatidic acid during in vitro maturation of bovine cumulus-oocyte complexes: cumulus expansion, glucose metabolism and expression of genes involved in the ovulatory cascade, oocyte and blastocyst competence.

BackgroundIn the cow, lysophosphatidic acid (LPA) acts as an auto-/paracrine factor, through its receptors LPAR1-4, on oocytes and cumulus cells during in vitro maturation (IVM). The aim of the present work was to determine the effect of LPA during IVM of bovine oocytes on: 1) oocyte maturation; 2) apoptosis of COCs; 3) expression of genes involved in developmental competence and apoptosis in bovine oocytes and subsequent blastocysts; 4) cumulus expansion and expression of genes involved in the ovulatory cascade in cumulus cells; 5) glucose metabolism and expression of genes involved in glucose utilization in cumulus cells; 6) cleavage and blastocyst rates on Day 2 and Day 7 of in vitro culture, respectively.MethodsCumulus-oocyte complexes (COCs) were matured in vitro in the presence or absence of LPA (10−5M) for 24h. Following maturation, we determined: oocyte maturation stage, cumulus expansion, COCs apoptosis and glucose and lactate levels in the maturation medium. Moreover, COCs were either used for gene expression analysis or fertilized in vitro. The embryos were cultured until Day 7 to assess cleavage and blastocyst rates. Oocytes, cumulus cells and blastocysts were used for gene expression analysis.ResultsSupplementation of the maturation medium with LPA enhanced oocyte maturation rates and stimulated the expression of developmental competence-related factors (OCT4, SOX2, IGF2R) in oocytes and subsequent blastocysts. Moreover, LPA reduced the occurrence of apoptosis in COCs and promoted an antiapoptotic balance in the transcription of genes involved in apoptosis (BAX and BCL2) either in oocytes or blastocysts. LPA increased glucose uptake by COCs via augmentation of GLUT1 expression in cumulus cells as well as stimulating lactate production via the enhancement of PFKP expression in cumulus cells. LPA did not affect cumulus expansion as visually assessed, however, it stimulated upstream genes of cumulus expansion cascade, AREG and EREG.ConclusionsSupplementation of the maturation medium with LPA improves oocyte maturation rates, decreases extent of apoptosis in COCs and sustains the expression of developmental competence related factors during oocyte maturation and subsequently affects gene expression profile at the blastocyst stage. We also demonstrate that LPA directs glucose metabolism toward the glycolytic pathway during IVM.

The importance of manganese in the cytoplasmic maturation of cattle oocytes: blastocyst production improvement regardless of cumulus cells presence duringin vitromaturation

Adequate dietary intake of manganese (Mn) is required for normal reproductive performance in cattle. This study was carried out to investigate the effect of Mn during in vitro maturation of bovine cumulus-oocyte complexes (COC) on apoptosis of cumulus cells, cumulus expansion, and superoxide dismutase (SOD) activity in the COC. The role of cumulus cells on Mn transport and subsequent embryo development was also evaluated. Early apoptosis decreased in cumulus cells matured with Mn compared with medium alone. Cumulus expansion did not show differences in COC matured with or without Mn supplementation. SOD activity was higher in COC matured with 6 ng/ml Mn than with 0 ng/ml Mn. Cleavage rates were higher in COC and denuded oocytes co-cultured with cumulus cells, either with or without Mn added to in vitro maturation (IVM) medium. Regardless of the presence of cumulus cells during IVM, the blastocyst rates were higher when 6 ng/ml Mn was supplemented into IVM medium compared with growth in medium alone. Blastocyst quality was enhanced when COC were matured in medium with Mn supplementation. The results of the present study indicated that Mn supplementation to IVM medium enhanced the 'health' of COC, and improved subsequent embryo development and embryo quality.

The effect of lysophosphatidic acid during in vitro maturation of bovine cumulus-oocyte complexes: cumulus expansion, glucose uptake and expression of expansion-related genes

The effect of lysophosphatidic acid during in vitro maturation of bovine cumulus-oocyte complexes: cumulus expansion, glucose uptake and expression of expansion-related genes

Amphiregulin co-operates with bone morphogenetic protein 15 to increase bovine oocyte developmental competence: effects on gap junction-mediated metabolite supply

This study assessed the participation of amphiregulin (AREG) and bone morphogenetic protein 15 (BMP15) during maturation of bovine cumulus-oocyte complexes (COCs) on cumulus cell function and their impact on subsequent embryo development. AREG treatment of COCs enhanced blastocyst formation and quality only when in the presence of BMP15. Expression of hyaluronan synthase 2 was enhanced by follicle-stimulating hormone (FSH) but not by AREG, which was reflected in the level of cumulus expansion. Although both FSH and AREG stimulated glycolysis, AREG-treated COCs had higher glucose consumption, lactate production and ratio of lactate production to glucose uptake. Autofluorescence levels in oocytes, indicative of NAD(P)H and FAD(++), were increased with combined AREG and BMP15 treatment of COCs. In contrast, these treatments did not alter autofluorescence levels when cumulus cells were removed from oocytes, even in the presence of other COCs, suggesting that oocyte-cumulus gap-junctional communication (GJC) is required. FSH contributed to maintaining GJC for an extended period of time. Remarkably, BMP15 was equally effective at maintaining GJC even in the presence of AREG. Hence, AREG stimulation of COC glycolysis and BMP15 preservation of GJC may facilitate efficient transfer of metabolites from cumulus cells to the oocyte thereby enhancing oocyte developmental competence. These results have implications for improving in vitro oocyte maturation systems.

9-cis Retinoic acid inhibits cumulus cell apoptosis during the maturation of bovine cumulus-oocyte-complexes1

Cumulus cell (CC) apoptosis is inversely correlated with embryonic development in vitro. Therefore, inhibition of CC apoptosis is important for proper embryonic development and quality. Retinoic acids (all-transRA and 9-cisRA) are natural components of retinoids, and 9-cisRA is the physiologically active metabolite of retinoic acid in vitro. During in vitro maturation, 9-cisRA enhances oocyte competence through multiple mechanisms affecting the oocyte and preimplantation embryo; however, the effect of 9-cisRA on CC apoptosis has yet to be elucidated. The aim of the present study was to evaluate the effect of 9-cisRA on CC apoptosis and to identify the molecular mechanism underlying that effect. Bovine slaughterhouse cumulus-oocyte complexes (COC) were matured in vitro in the absence or presence of 5 nM 9-cisRA. Cumulus cells were collected from immature and matured COC for the detection of apoptosis and gene expression analysis. Results showed that 9-cisRA reduced the number of apoptotic CC by about 2.7 fold (P < 0.023), compared with control. However, apoptosis is rare in CC of immature COC (0.01% ± 0.001). Transcripts involved in the caspase cascade were down-regulated upon exposure to 9-cisRA, including tumor necrosis factor alpha (TNF-α, 11.1 fold, P < 0.001), tumor necrosis factor alpha receptor 1 (TNFR1, 2.3 fold, P < 0.01), caspase 9 (CASP9, 2.0 fold, P < 0.031), caspase 8 (CASP8, 2.2 fold, P < 0.012), and caspase 3 (CASP3, 2.1 fold, P < 0.006), while antiapoptotic B-cell lymphoma 2 (BCL2) transcript was increased (3.1 fold, P < 0.004), compared with control. In addition, 9-cisRA inhibited mitogen activated protein kinase mRNA expression in CC, including extracellular signal-regulated kinase 1/2 (ERK1, 2.7 fold, P < 0.02; ERK2, 2.7 fold, P < 0.03), and c-Jun N-terminal kinase (JNK, 1.6 fold, P < 0.044), as well as the activator protein-1 (AP1) family members c-jun (1.6 fold, P < 0.041) and c-fos (2.0 fold, P < 0.06). The transcript abundances of TNF-α, TNFR1, CASP9, CASP8, CASP3, ERK1, ERK1, JNK, and BCL2 were increased, while c-fos and c-jun mRNA expression was decreased in the matured CC. On the basis of the data, we suggest that 9-cisRA inhibits CC apoptosis during in vitro maturation of bovine COC.

Effect of α-tocopherol on in vitro maturation of bovine cumulus-oocyte complexes

This study determined: the effects of α-tocopherol on apoptotic and necrotic levels of cumulus cells after in vitro maturation of bovine oocytes; whether exposure to α-tocopherol facilitates the development of bovine enclosed oocytes to metaphase II; and the effects of this antioxidant on apoptotic and necrotic levels of granulosa cells cultured in vitro. In conclusion, supplementation with α-tocopherol on in vitro maturation of bovine oocytes has a detrimental effect on the ability of oocytes to reach metaphase II, increasing the number of apoptotic and necrotic cumulus cells of bovine cumulus oocyte complexes (COC). This antioxidant showed a slight improvement in the viability of cultured granulosa cells at a concentration of 100 µM. Key words: Bovine, oocyte maturation in vitro, antioxidant, α-tocopherol

Expression of the vascular endothelial growth factor and its receptors and effects of VEGF during in vitro maturation of bovine cumulus-oocyte complexes (COC).

A complete VEGF system consisting of the ligand and two of its receptors has been detected for the first time in the bovine cumulus-oocyte-complex (COC). In the course of a 24 hr in vitro maturation procedure (IVM), expression of the smaller VEGF transcripts and their specific receptors flt and flk changed remarkably in a time-dependent manner as observed by RT-PCR. The transcript concentrations of VEGF declined within 24 hr of culture, whereas both receptor mRNAs were found enriched between 6 and 12 hr of IVM. In the follicular fluid of growing ovarian follicles, immunoreactive VEGF, measured by RIA, increased significantly, reaching highest concentrations immediately before ovulation of the oocyte. The immunohistochemical localization of VEGF in bovine COCs revealed strong signals within the cumulus cell complex clearly extending beyond the oocyte cytoplasm at the beginning of in vitro maturation. After 24 hr, IVM immunoreactive VEGF disappeared remarkably from cumulus cells and the oocyte cytoplasm. An exogenous application of VEGF at the beginning of a 24 hr IVM significantly improved cleavage rates of zygotes and their development into bovine embryos showing obvious synergistic effects in combination with FSH, when compared with untreated control embryos. In addition, the number of blastomeres in deriving blastocysts increased after VEGF supplementation. These results indicate a functional VEGF system controlling important events beside the known angiogenetic effect during in vivo and in vitro maturation of the bovine COC, possibly affecting the early embryonic viability.

Effects of ethanol and dimethylsulphoxide on nuclear and cytoplasmic maturation of bovine cumulus-oocyte complexes.

The influence of small doses of ethanol or dimethylsulphoxide (DMSO) on in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COC) was examined, either after spontanous maturation or after inhibition of meiosis with 6-dimethylaminopurine (6-DMAP) or 3-isobutyl-1-methylxanthine (IBMX). Subsequent to IVM for 23 hr in semidefined serum-free Earle's TCM199 medium, nuclear maturation was assessed cytogenetically, while the combined cytoplasmic and nuclear maturation was measured indirectly by the oocytes' ability to undergo fertilization and further embryonic development. Embryo development was followed until the blastocyst stages at day 9 after insemination. Neither spontanous nuclear maturation nor cleavage was compromised by IVM in </=1% (v/v) ethanol or </=1% (v/v) DMSO, nor was the frequency of polyspermy altered. However, IVM in 0.3% or 1% (v/v) ethanol or in 0.4 or 1% (v/v) DMSO negatively affected blastocyst formation, compared to 0% in the control groups (22% and 23% vs. 34%, P < 0.0001, and 29% and 22% vs. 34%, P < 0.01, respectively), whereas the speed of blastocyst formation, assessed as the D7/D9 blastocyst proportion, was not compromised. In oocytes meiotically inhibited with 2 mM 6-DMAP, the presence of ethanol (0. 5%, 1%, and 2% [v/v]) induced germinal vesicle breakdown in a dose-dependent manner (32%, 45%, and 68%, vs. 22%, P < 0.0001), however, the oocytes exhibited no further meiotic progression. In oocytes inhibited with 1 and 2 mM IBMX, the presence of ethanol (0. 5%, 1%, and 2% [v/v]) significantly (P < 0.05) enhanced the inhibitory effect in a dose-dependent manner by reducing the proportion of the mature (AI-MII) stages (77%, 68%, and 56% vs. 79%, and 33%, 29%, and 18% vs. 39%, respectively). It is concluded that even small doses of ethanol or DMSO can cause profound negative effects on bovine in vitro maturation and subsequent embryo development.

Effects of ethanol and dimethylsulphoxide on nuclear and cytoplasmic maturation of bovine cumulus-oocyte complexes

The influence of small doses of ethanol or dimethylsulphoxide (DMSO) on in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COC) was examined, either after spontanous maturation or after inhibition of meiosis with 6-dimethylaminopurine (6-DMAP) or 3-isobutyl-1-methylxanthine (IBMX). Subsequent to IVM for 23 hr in semidefined serum-free Earle's TCM199 medium, nuclear maturation was assessed cytogenetically, while the combined cytoplasmic and nuclear maturation was measured indirectly by the oocytes' ability to undergo fertilization and further embryonic development. Embryo development was followed until the blastocyst stages at day 9 after insemination. Neither spontanous nuclear maturation nor cleavage was compromised by IVM in ≤1% (v/v) ethanol or ≤1% (v/v) DMSO, nor was the frequency of polyspermy altered. However, IVM in 0.3% or 1% (v/v) ethanol or in 0.4 or 1% (v/v) DMSO negatively affected blastocyst formation, compared to 0% in the control groups (22% and 23% vs. 34%, P < 0.0001, and 29% and 22% vs. 34%, P < 0.01, respectively), whereas the speed of blastocyst formation, assessed as the D7/D9 blastocyst proportion, was not compromised. In oocytes meiotically inhibited with 2 mM 6-DMAP, the presence of ethanol (0.5%, 1%, and 2% [v/v]) induced germinal vesicle breakdown in a dose-dependent manner (32%, 45%, and 68%, vs. 22%, P < 0.0001), however, the oocytes exhibited no further meiotic progression. In oocytes inhibited with 1 and 2 mM IBMX, the presence of ethanol (0.5%, 1%, and 2% [v/v]) significantly (P < 0.05) enhanced the inhibitory effect in a dose-dependent manner by reducing the proportion of the mature (AI-MII) stages (77%, 68%, and 56% vs. 79%, and 33%, 29%, and 18% vs. 39%, respectively). It is concluded that even small doses of ethanol or DMSO can cause profound negative effects on bovine in vitro maturation and subsequent embryo development. Mol. Reprod. Dev. 55:438–445, 2000. © 2000 Wiley-Liss, Inc.

Low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine cumulus-oocyte complexes

The effects of carbohydrates on meiotic maturation and ATP content of bovine oocytes under low oxygen tension (5%) were investigated. Furthermore, the developmental competence or intracellular H(2)O(2) contents of the oocytes matured under 5% or 20% O(2) was assessed. In vitro maturation of bovine cumulus-oocyte complexes was performed in synthetic oviduct fluid (SOF) containing 20 amino acids and hormones (SOFaa). The proportion of the oocytes that matured to the metaphase II stage in SOFaa containing 1.5 mM glucose, 0.33 mM pyruvate, and 3.3 mM lactate under 5% O(2) was dramatically lower than that of oocytes matured under 20% O(2) (P < 0.01). Similarly, the ATP content of the oocytes that matured under 5% O(2) was much lower than that of oocytes matured under 20% O(2) (P < 0.05). Under 5% O(2) the proportion of metaphase II oocytes increased with increasing glucose concentration (0-20 mM) in SOFaa without pyruvate or lactate. In addition, the ATP content of oocytes cultured in 20 mM glucose was higher (P < 0.05) than that of oocytes cultured in 1. 5 mM glucose. Two glucose metabolites (pyruvate and lactate) and a nonmetabolizable glucose analog (2-deoxy-glucose), however, had no noticeable effects on meiotic maturation under 5% O(2). These results suggest that ATP production under 5% O(2) is not dependent on the TCA cycle. Addition of iodoacetate, a glycolytic inhibitor, to SOFaa containing 20 mM glucose significantly reduced (P < 0.01) the proportion of metaphase II and ATP content. Moreover, the proportion of the development to the blastocyst stage of oocytes matured under 5% O(2) was higher (P < 0.05) than that of oocytes matured under 20% O(2). H(2)O(2) contents of oocytes matured under 5% O(2) was lower (P < 0.05) than that of oocytes matured under 20% O(2). The results of the present study demonstrate that glucose plays important roles in supporting the completion of meiotic maturation in bovine cumulus-oocyte complexes under low oxygen tension and that low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine oocytes.

Demonstration of a suppressive effect of inhibin alpha-subunit on the developmental competence of in vitro matured bovine oocytes.

Changes in intrafollicular concentrations of different forms of inhibin (free alpha-subunits and alpha beta dimers) occur during follicle development and may influence the oocyte maturation process. The aim of this study was to investigate the effects of inhibin A and free alpha-subunit (pro-alpha C) isolated from bovine follicular fluid on maturation of bovine cumulus-oocyte complexes, as reflected by their competence for embryo development after in vitro fertilization. Bovine cumulus-oocyte complexes were isolated from ovaries obtained from an abattoir and were cultured for 22-24 h at 38.5 degrees C in TCM-199 medium supplemented with 10% oestrous cow serum, pregnant mares' serum gonadotrophin (2.5 iu ml-1) and either inhibin A (0, 0.2 and 1.0 microgram ml-1) or pro-alpha C (0, 2 and 10 micrograms ml-1). Neither inhibin A nor free alpha-subunit affected the cleavage rate of cumulus-oocyte complexes after fertilization (approximately 60%). Inhibin A reduced the proportion of cleaved oocytes reaching the eight-cell stage by 19% (P < 0.05), but did not affect the yield of blastocysts. However, pro-alpha C decreased the proportion of cleaved oocytes that reached the eight-cell (25%; P < 0.05) and blastocyst (28%; P < 0.05) stages. In addition, a negative correlation (r = -0.55, P < 0.001) was found between concentrations of total immunoreactive (ir) alpha-inhibin (measured by radioimmunoassay) produced by untreated control cumulus-oocyte complexes and their post-cleavage development to the blastocyst stage. In a second experiment, mouse monoclonal antibodies (20 micrograms ml-1) against two different regions of the inhibin alpha-subunit precursor (pro-region and alpha C fragment) were tested for their ability to neutralize endogenous inhibin alpha-subunit-related molecules produced by cumulus cells; control cumulus-oocyte complexes were treated with normal mouse IgG (20 micrograms ml-1). Although the cleavage rate was not affected, the yield of blastocysts was significantly higher in the presence of mouse monoclonal antibodies to both pro-alpha (77% increase; P < 0.05) and alpha C (48% increase; P < 0.05). None of the treatments tested affected endogenous production of activin-A or follistatin by cumulus-oocyte complexes. Overall, these results indicate that the inhibin alpha-subunit (pro-alpha C) has an inhibitory role in oocyte maturation which is independent of the modulatory effects of activin and follistatin.

Growth factors and components for extracellular proteolysis are differentially expressed during in vitro maturation of bovine cumulus-oocyte complexes.

In the present study, the time-dependent collagenolytic potential and mRNA transcription of extracellular matrix (ECM)-degrading components, transforming growth factor beta1 (TGFbeta1), and both basic fibroblast growth factor (bFGF) and FGF receptors (FGFR) in bovine cumulus-oocyte complexes (COCs) were investigated during 24 h of in vitro maturation (IVM). COCs were collected from 2- to 6-mm follicles, cultured in maturation medium, and sequentially removed at 3-h intervals for analysis. From 285 oocytes matured under these conditions, 114 (40.0%) developed to blastocysts on Day 9 after fertilization. Gelatin zymograms revealed protease activity at about 55 kDa for COCs matured for at least 3 h and two additional proteolytic zones at about 70 kDa after at least 9 h of IVM. The mRNAs of ECM-degrading components urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI1), matrix metalloproteinase 1 (MMP1), and tissue inhibitor of metalloproteinases 1 (TIMP1), as well as TGFbeta1, bFGF, and FGFR, were detected during IVM in a factor-specific pattern: all transcript levels found at COC 0 generally increased after 3 h of maturation and either remained high or decreased thereafter. On the basis of the chosen reverse transcription-polymerase chain reaction technique, one could suggest relative higher mRNA concentrations for TIMP1, PAI1, and both growth factors compared to uPA, MMP1, and FGFR. Our results suggest a finely tuned extracellular proteolysis during IVM of bovine COCs, for which the concerted action of modulating growth factors like bFGF and TGFbeta1 may be essential.

Control of in vitro maturation of bovine cumulus-oocyte complex by preovulatory granulosa cells.

The present study was designed to investigate 1) the influence of the secretions of follicular cells on the in vitro maturation of bovine cumulus-oocyte complexes (COCs) and 2) the origin of the factors controlling the metabolic function of cumulus cells during the preovulatory period. Preovulatory granulosa cells were collected from synchronized heifers either before or 7-9 hr after the luteinizing hormone (LH) surge, and their secretions were recovered after a 3 hr incubation. Follicular fluids (FFs) originating from the same follicles and sera from the same animals were also collected. The effects of FFs, sera, and secretions of granulosa cells on COC metabolism were compared during 24 hr of culture. FF stimulated cumulus expansion, progesterone secretion, and overall protein synthesis by COCs but decreased the amount of a major protein of 28 kDa. The time at which FF was collected influenced both cumulus expansion and protein synthesis by COCs. The effects of FF on COC metabolism were detected at the lowest protein concentration studied (0.073 mg/ml) and could be mimicked with serum, but only at a protein concentration 100-fold higher. The inhibitory effect of FF and serum on the amount of the 28 kDa protein was reproduced with the secretions of granulosa cells, acting at protein concentrations five- and 500-fold lower, respectively. However, the secretions of granulosa cells enhanced slightly cumulus expansion and had no effect on progesterone secretion and overall protein synthesis by COCs. These results suggest that COC metabolism is influenced both by endocrine and by local factors secreted by granulosa cells in response to gonadotropins. The paracrine control of COC metabolism by preovulatory granulosa cells could be exerted not only via intercellular contacts but also via substances secreted in FF.

Fertilizability and subsequent developmental ability of bovine oocytes matured in medium containing epidermal growth factor (EGF)

We have previously shown that epidermal growth factor (EGF) is capable of promoting maturation of bovine cumulus-oocyte complexes in chemically defined serum-free medium. In this study, fertilizability and subsequent developmental capacity of bovine oocytes matured in EGF-containing medium were evaluated. Fetal bovine serum (FBS, 10%) and EGF at 10 ng/ml in Dulbecco's modified Eagles medium with Ham's nutrient mixture F-12 ( DME F12 ) significantly increased the rate of formation of two pronuclei compared with the rate obtained from DME-F12 alone (P<0.05). Early embryonic development was assessed during 48 h in culture. Data were evaluated in terms of cleavage and four- to eight-cell formation. Oocytes matured in 10 ng/ml EGF showed significantly higher rates of cleavage (P<0.01) and four- to eight-cell formation than did oocytes matured in control medium (P<0.05). Bovine oocytes matured in the presence of EGF can be normally fertilized and can cleave and develop in vitro up to the eight-cell stage.