Kinetics Of Nuclear Maturation Research Articles

Nuclear maturation kinetics and in vitro fertilization of immature bovine oocytes injected into pre-ovulatory follicles

The maturation kinetics and in vitro fertilization of immature bovine oocytes injected by the intra-follicular oocyte injection (IFOT) technique into pre-ovulatory follicles of previously synchronized cows were evaluated. In Experiment 1, grade I, II and III cumulus-oocyte complexes (COCs) were randomly distributed to one of three Groups: Matvitro22 (COCs matured in vitro for 22 h), MatFol20 and MatFol28 (COCs matured in vivo after being injected into a pre-ovulatory follicle of previously synchronized cows for 19.8 ± 0.1 h and 28.3 ± 0.1 h, respectively). Cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at the time of IFOT in the MatFol20 Group or 10 h after IFOT in the MatFol28 Group. MatFol20 and MatFol28 COCs were aspirated approximately 20 h after the LH injection for nuclear maturation kinetics and recovery rate assessment. In Experiment 2, grade I, II, and III COCs were randomly distributed into two Groups: Matvitro22 Group, COCs were matured and fertilized in vitro, and MatFol20 Group, COCs were matured as in the MatFol20 Group in Experiment 1, but COCs were fertilized in vitro. Putative zygotes were classified as fertilized, unfertilized or polyspermic. In Experiment 1, the recovery rate was lower (P < 0.001) in the MatFol20 Group (52.9%, 91/172) compared with MatFol28 (72.9%, 113/155). Rate of oocytes in germinal vesicle stage, metaphase I, anaphase I and telophase I were similar among Groups. However, oocytes matured in vivo for 28.3 h had lower rate of metaphase II (P = 0.001) and greater rates of degenerated (P = 0.001) and parthenogenetically activated (P = 0.001) oocytes. In experiment 2, the rates of polyspermy and degenerated were similar between Groups. However, the rate of fertilized oocytes was greater (P = 0.05) in oocytes in the MatFol20 Group. It is concluded that oocyte in vivo maturation for 19.8 h after IFOT does not compromise the nuclear maturation kinetics and increases in vitro fertilization rates. However, the extra 10 h of intra-follicular incubation time decreased oocyte viability.

169 Nuclear Maturation Kinetics and In Vitro Fecundation of Immature Bovine Oocytes Injected into Preovulatory Follicles

The objective was to evaluate in vitro nuclear maturation and fecundation kinetics of oocytes injected into preovulatory follicles of synchronized cows using the intra-follicular oocyte injection (IFOI) technique. In experiment 1, 438 immature abattoir-bovine cumulus–oocyte complexes (COC) of grades I, II, and III were randomly allocated to 1 of 3 groups: Matvitro (n = 111), COC matured in vitro for 22 h; Matvivo20 (n = 172) and Matvivo30 (n = 155), 30 oocytes were injected into each preovulatory follicle of pre-synchronized recipients. In Matvivo20, oocytes were matured for 19.8 ± 0.1 h and in Matvivo30, for 28.3 ± 0.1 h. All cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at IFOI (Matvivo20) or 10 h after IFOI (Matvivo30). Oocytes from Matvivo20 and Matvivo30 were aspirated 20 h after LH injection for assessment of oocyte maturation and recovery rates. Oocytes were evaluated according to maturation kinetics as germinal vesicle, metaphase I, anaphase I, telophase I, metaphase II, parthenogenetically activated, and degenerated (chromosomal aberrations, presence of diffuse or indefinite chromatin). In experiment 2, immature abattoir-bovine COC (n = 202) of grades I, II, and III were randomly distributed into 2 groups: Matvitro (n = 103), COC were matured and fertilized in vitro; Matvivo (n = 99), same as Matvivo20 protocol, and COC fertilized in vitro. Presumptive zygotes were evaluated as fertilized, unfertilized, or polyspermic. Statistical analyses were performed by the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC, USA). Recovery rate was lower (P &lt; 0.001) in Matvivo20 (52.9%, 91/172) compared with Matvivo30 (72.9%, 113/155). Germinal vesicle (P = 0.94), metaphase I (P = 0.98), anaphase I (P = 0.99), and telophase I (P = 0.20) rates were similar. However, there were differences in metaphase II [Matvitro: 81.0% (90/111)a, Matvivo20: 74.5% (35/47)a, and Matvivo30: 41.6% (32/77)b; P = 0.001], degenerate [Matvitro: 5.4% (6/111)c, Matvivo20: 21.3% (10/47)b and Matvivo30: 48.1% (37/77); P = 0.001] and parthenogenetically activated [Matvitro: 0.0% (0/111)b, Matvivo20: 0.0% (0/47)b and Matvivo30: 9.1% (7/77)a; P = 0.001]. Polyspermic (P = 0.18) and abnormal (P = 0.98) rates were similar. However, there was a higher rate (P = 0.05) of fertilized oocytes in Matvivo (60.6%, 60/99) than in Matvitro (46.6%, 48/103). In conclusion, oocyte maturation in vivo after IFOI for 20 h does not alter maturation kinetics and increases in vitro oocyte fertilization capacity. However, the 10-h increase in intra-follicular oocyte permanence decreased the proportion of viable oocytes. Thus, the oocyte maturation phase is not the limiting causative factor for the low IFOI-embryo production rates.

Bovine in vitro embryo production: the effects of fibroblast growth factor 10 (FGF10).

In an attempt to improve in vitro embryo production, we investigated the effect of fibroblast growth factor 10 (FGF10) during in vitro maturation on the developmental capacity of bovine oocytes. Cumulus-oocyte complexes (COCs) were aspirated from follicles of 3-8mm diameter. After selection, the COCs were matured in medium with or without 0.5ng/mL of FGF10. The effect of FGF10 during in vitro maturation (IVM) on nuclear maturation kinetics and expansion of the cumulus cells was investigated. Oocyte competence was assessed by the production and development speed of embryos and the relative expression of genes associated with embryo quality. FGF10 delayed the resumption of meiosis from 8h onwards, but did not affect the percentage of oocytes reaching metaphase II, nor did it increase cumulus expansion at 22h of maturation. We found no difference between treatments regarding embryo production, developmental speed, and gene expression. In conclusion, the presence of FGF10 during IVM had no effect on embryo production, developmental speed, and gene expression.

The Effect of Cilostamide on Gap Junction Communication Dynamics, Chromatin Remodeling, and Competence Acquisition in Pig Oocytes Following Parthenogenetic Activation and Nuclear Transfer1

In the pig, the efficiency of in vitro embryo production and somatic cell nuclear transfer (SCNT) procedures remains limited. It has been suggested that prematuration treatments (pre-IVM) based on the prolongation of a patent, bidirectional crosstalk between the oocyte and the cumulus cells through gap junction mediate communication (GJC), with the maintenance of a proper level of cAMP, could improve the developmental capability of oocytes. The aim of this study was to assess: 1) dose-dependent effects of cilostamide on nuclear maturation kinetics, 2) the relationship between treatments on GJC functionality and large-scale chromatin configuration changes, and 3) the impact of treatments on developmental competence acquisition after parthenogenetic activation (PA) and SCNT. Accordingly, cumulus-oocyte complexes were collected from 3- to 6-mm antral follicles and cultured for 24 h in defined culture medium with or without 1 μM cilostamide. GJC functionality was assessed by Lucifer yellow microinjection, while chromatin configuration was evaluated by fluorescence microscopy after nuclear staining. Cilostamide administration sustained functional coupling for up to 24 h of culture and delayed meiotic resumption, as only 25.6% of cilostamide-treated oocytes reached the pro-metaphase I stage compared to the control (69.7%; P < 0.05). Moreover, progressive chromatin condensation was delayed before meiotic resumption based upon G2/M biomarker phosphoprotein epitope acquisition using immunolocalization. Importantly, cilostamide treatment under these conditions improved oocyte developmental competence, as reflected in higher blastocyst quality after both parthenogenetic activation and SCNT.

A simple and high-throughput method to assess maturation status of bovine oocytes: Comparison of anti-lamin A/C-DAPI with an aceto-orcein staining technique

A precise, accurate, nonambiguous and high-throughput method is required to assess nuclear maturation of mammalian oocytes. The objectives of this study were to compare the efficiency and ease of use of a simplified fluorescence imaging (anti-lamin A/C and 4′,6-diamidino-2-phenylindole [DAPI]) technique to the existing technique (aceto-orcein staining) for the evaluation of nuclear maturation of bovine oocytes, and to determine the kinetics of bovine oocyte maturation using an anti-lamin A/C-DAPI technique. In Experiment 1, oocytes were matured in vitro and stained with aceto-orcein and anti-lamin A/C-DAPI staining techniques. The proportions of oocytes lost during procedures and those that could not be classified (because of ambiguous morphology) during evaluation were lower (P < 0.0001) in oocytes stained with anti-lamin A/C-DAPI (9% and 2%) than those stained with aceto-orcein (31% and 13%), respectively. Anti-lamin A/C-DAPI was a quick procedure which could be completed within 7 h after completion of the maturation (compared with > 24 h for the aceto-orcein method). Furthermore, > 200 oocytes could be stained in one batch with anti-lamin A/C-DAPI technique. In Experiment 2, nuclear maturation kinetics of bovine oocytes at various time intervals (0, 6, 12, and 22 h) during in vitro maturation (IVM) was evaluated using the anti-lamin A/C-DAPI technique. Germinal vesicle, germinal vesicle breakdown, metaphase I, and metaphase II oocytes were predominant at 0, 6, 12, and 22 h of IVM, respectively. We concluded that the anti-lamin A/C-DAPI was an efficient and simple technique for nonambiguous evaluation of nuclear maturation status of large numbers of oocytes in a short interval.

The effects of macromolecular and serum supplements and oxygen tension during bovine in vitro procedures on kinetics of oocyte maturation and embryo development

Aiming to standardize in vitro production of bovine embryos and to obtain supplements to replace serum in culture media, this study evaluated the nuclear maturation kinetics and embryonic development in bovine after in vitro maturation (IVM) and culture (IVC) with several macromolecules (animal origin: bovine serum albumin (BSA), fetal calf serum (FCS); synthetic: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), Ficoll, and Knockout) at two oxygen tensions (20% and 5% O(2)). Regarding nuclear kinetics, neither the presence of the expected stage (metaphase I, transition anaphase to telophase, and metaphase II) at each evaluation moment (6, 18, and 24h after IVM, respectively) nor the accelerated polar body emission (at 18h after IVM) related developmental competence to blastocyst stage when different supplements were compared. Independently of supplement, cleavage rates at 20% O(2) (61.6-79.2%) were higher than at 5% O(2) (38.9-58.7%). At 20% O(2), higher blastocyst and hatching rates, respectively, were obtained in treatments BSA, FCS, Knockout, and control group (IVM with FCS and IVC with BSA + FCS, 14.0-23.5% and 6.8-15.4%) in comparison to PVA, PVP, and Ficoll (0%). The same was observed at 5% O(2) for blastocyst rates with BSA, FCS, Knockout, and control (5.4-16.8%) and for hatching rates with BSA, FCS, and control (2.0-11.1%). We can conclude that producing bovine embryos at 20% O(2) during the entire IVP process resulted in higher developmental rates than at 5% O(2). In addition, while defined macromolecules PVA, PVP, and Ficoll were not suitable for embryonic development, the synthetic serum Knockout was able to replace serum and albumin for IVP in bovine at 20% O(2).

Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine

Cyclin dependent kinase inhibitors (CDKIs) may be used for pre-maturation culture, but can accelerate nuclear maturation. The aim of the present research was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at lesser than typically used concentrations on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 μM BLI (B) or 6.25 μM BLI + 12.5 μM ROS (BR) in TCM-199 for 24 h. Oocytes were subsequently submitted to in vitro maturation (IVM) in TCM-199 + 0.5 μg/ml FSH, 50 μg/ml LH and 10% FCS for another 24 h, during which oocytes were fixed every 3 h. In Experiment 2, oocytes were submitted to 24 h pre-maturation treatments, with the inhibitors being diluted in TCM-199 or DMEM. IVM lasted 21 h in the culture media DMEM + 0.5 μg/ml FSH, 50 μg/ml LH, 5% FCS and 50 ng/ml EGF. After IVM, oocytes from all groups were fertilized in vitro. Oocytes and sperm (2 × 10 6 sperm cells/ml) were co-cultured for 18 h. Embryos were co-cultured with granulosa cells in CR2aa for 8 days. All cultures were in droplets under oil, at 38.5 °C and 5% CO 2 in air. In both experiments, control oocytes (C) were submitted only to IVM. In Experiment 1, at 0 h, C and B oocytes were all (100%) at the germinal vesicle stage (GV) of development. BR had fewer GV oocytes (89%, P < 0.05). After 3 h IVM, B and BR had fewer oocytes in GV (84.7 and 79.6%, P > 0.05) than C (100%, P < 0.05). At 12 h, most oocytes were at intermediate stages (metaphase to telophase I) in all groups (∼80%, P > 0.05). After 21 (77–89%) and 24 h (85–95%), all groups had similar metaphase II (MII) rates of development ( P > 0.05). In Experiment 2, cleavage (79–84%, P > 0.05) and Day 7 blastocyst rates (26–36%, P > 0.05) were similar. After 8 days, the group pre-matured with BR in DMEM had lesser blastocyst rates of development (32.3%) lower than C (40.1%, P < 0.05). The other groups were similar to C (35–38%, P > 0.05). Hatching rates were similar (10–15%, P > 0.05) as were total cell numbers (141–170). In conclusion, BR is less effective in maintaining meiosis block; B and BR accelerate meiosis resumption; and use of pre-maturation medium may affect developmental rates.

Influence of the duration of in vitro maturation and gamete co-incubation on the efficiency of in vitro embryo development in Italian Mediterranean buffalo ( Bubalus bubalis)

The aim of this study was to investigate the effect of the duration of oocyte in vitro maturation (IVM) and gamete co-incubation on the in vitro embryo (IVEP) production efficiency in River buffalo. In Experiment 1, abattoir-derived cumulus oocyte complexes were fixed at 0, 3, 6, 9, 12, 15, 18, 21 and 24 h after the start of in vitro maturation to study the kinetics of nuclear maturation. In Experiment 2, cumulus oocyte complexes were fertilized in vitro following in vitro maturation for 18, 21, 24, 27 or 30 h. After 20 h of gamete co-incubation, presumptive zygotes were denuded and cultured in vitro in synthetic oviduct fluid (SOF) medium. In Experiment 3, following in vitro maturation and fertilization, presumptive zygotes were removed from fertilization drops at 8, 12, 16 and 20 h post-insemination (pi) and placed in culture as described above. Representative samples of oocytes were fixed at 4, 8, 12, 16 and 20 h to evaluate the sperm penetration rate and the incidence of polyspermy at different co-incubation times. The main conclusions of the study are that: (1) the majority of buffalo oocytes accomplish nuclear maturation between 21 and 24 h after the start of in vitro maturation; (2) both cleavage and blastocyst rates linearly decrease with increasing duration of in vitro maturation (from 18 to 30 h); (3) sperm–oocyte incubation for at least 16 h is required for maximum blastocyst yields.

Effect of sodium nitroprusside, a nitric oxide donor, on the in vitro maturation of bovine oocytes

Nitric oxide (NO) is a highly reactive free radical involved in intra- and intercellular signaling in various stages of reproduction. The objective of the present study was to evaluate the effect of the addition of sodium nitroprusside (SNP), a NO donor, on nuclear and cytoplasmic in vitro maturation of bovine oocytes. Analysis of variance was conducted and the means were compared by t test at a level of 5%. Low (10 −7 and 10 −9 M) and intermediate (10 −5 M) concentrations of SNP had no significant effect on nuclear maturation, however, when a greater concentration of SNP (10 −3 M) was added, oocytes remained in metaphase I (MI) after 24 h culture ( P < 0.05) and did not show cumulus expansion. To evaluate if this effect was reversible and if a retardation or inhibition had occurred in the progression from MI to MII, oocytes were cultured in presence of 10 −3 M of SNP for 24 h followed by culture for an additional 24 h in medium with or without SNP. After 48 h, the oocytes remained in MI even when the medium was changed at 24 h with or without SNP. The kinetics of nuclear maturation was assessed to evaluate if there had been or not a retardation in the progression of meiosis with the concentration of 10 −3 M SNP. This concentration delayed germinal vesicle breakdown (VGBD) at 8 h of culture ( P < 0.05), and at 12 h there was no significant difference between the control and the treated group. The concentrations that did not induce alterations in nuclear maturation were evaluated for cytoplasmic maturation. The concentration of 10 −5 M improved the percentage of peripheral cortical granules ( P < 0.05), and significantly increased the percentage of blastocysts. These results demonstrate that SNP at greater concentrations (10 −3 M) has a cytotoxic effect, but at intermediate (10 −5 M) concentrations it increases blastocyst rates. NO exhibits a dual effect on bovine oocytes, inhibits (10 −3 M of SNP) nuclear and cytoplasmic maturation or stimulates (10 −5 M of SNP) cytoplasmic maturation, depending on concentration in the culture medium.

The effect of glucose on the progression of the nuclear maturation of pig oocytes

The progression of the nuclear maturation of oocytes is a useful marker for the estimation of the subsequent developmental competence of oocytes. In this study, we examined the effect of energy substrates in an in vitro maturation medium on the progression of the nuclear maturation of oocytes. In experiment 1, the supplementation of the maturation medium with 0, 5 and 10 mM of glucose lead to increase in the total cell number of the blastocysts. In experiments 2 and 3, the maturation phase was divided into two stages (germinal vesicle (GV) stage: 0–20 h and nuclear maturation stage: 20–44 h), and the effects of glucose or pyruvate added at each stage on the kinetics of nuclear maturation were examined. The addition of glucose at the nuclear maturation stage rather than at the GV stage of maturation effected greater acceleration in the progression of nuclear maturation. However, the addition of pyruvate at both stages had the same effect on the progression of nuclear maturation was the same. In addition, when glucose was added to the medium containing pyruvate, an additive effect on the progression of nuclear maturation was observed (experiment 4). In experiment 5, the inhibitors of glucose-6-phosphate dehydrogenase (G6PD), dehydroepiandrosterone (DHEA) and 6-aminonicotinamide (6-AN) decreased the rate of the final maturation of oocytes and reduced the difference between the rates of the final maturation of oocytes cultured with glucose and those cultured with pyruvate. In the experiment 6, when the activator of G6PD, brilliant cresyle blue (BCB), was added to the maturation medium, the progression of nuclear maturation was significantly accelerated. The results of this study suggested that in addition to the role of an energy substrate, glucose or its metabolites play a role in nuclear maturation. This role was more pronounced at the second stage of maturation (transition from GV breakdown (GVBD) to M2), probably due to the metabolism of glucose via the pentose phosphate pathway (PPP) rather than the glycolysis pathway.

Effect of concentration and exposure period to butyrolactone I on meiosis progression in bovine oocytes

The effect of concentration and exposure period of bovine oocytes to butyrolactone I (BLI) on meiotic block and in vitro maturation (IVM) kinetics was studied. In experiment 1, all oocytes were at germinal vesicle stage (GV), after 6h in culture with 0, 50 and 100µM BLI. After 12h, all oocytes cultured with 50 and 100µM BLI remained in GV. After 24h, less oocytes were in GV with 50µM (82%) than with 100µM BLI (99%, P&lt;0.05). In experiment 2, after 6h IVM, 93% of control oocytes (IVM only) were in GV, while treated oocytes (100µM BLI for 6, 12 or 24h prior to IVM) showed less oocytes in GV with increased exposure period to BLI prior to IVM (83 and 73%, for 6h and 12h, P&lt;0.05). For a 24h inhibition, GV rates were similar to 12h (70%, P&gt;0.05). After 18h IVM, metaphase II (MII) rates were similar for all groups (76-81%). In experiment 3, after 6h IVM, 74% of treated oocytes (50 or 100µM BLI for 12h) were in GV. This rate was lower than for control oocytes (97.3%, P&lt;0.05). After 18h IVM more oocytes (~80%, P&gt;0.05) were in MII with BLI than for control (73%, P&lt;0.05). Shorter culture periods require lower BLI concentration for meiotic block; initial nuclear maturation kinetics of oocytes cultured with BLI is accelerated, and this is affected by culture period but not by drug concentration.

344 FOLLICULAR FLUIDS COLLECTED FROM LARGE FOLLICLES IMPROVED THE PROGRESSION OF IN VITRO NUCLEAR MATURATION OF PIG OOCYTES

The present study examined the effects of supplementation of in vitro maturation medium with porcine follicular fluids (pFFs) on the progression of nuclear maturation, subsequent fertilization, and development of pig oocytes. The pFFs were collected from follicles of different diameters: small follicles (SFs, 3–4 mm in diameter) and large follicles (LFs, 5-6 mm). In experiment 1, oocytes were collected from follicles 3–6 mm in diameter. The oocytes were cultured in maturation medium (NCSU-37) supplemented (10% v/v) with pFF from SFs (SpFF) or LFs (LpFF); the kinetics of nuclear maturation, fertilization rate, and developmental competence of oocytes were evaluated. In experiment 2, oocytes were collected from SFs or LFs, and the effects of supplementation of SpFF and LpFF on the nuclear maturation of these oocytes were examined. In experiment 3, each pFF was denaturated by heat treatment (60�C for 30 min) or trypsin treatment (200 �g mL-1), and the effects of these pFFs on the nuclear maturation of pig oocytes were examined. In experiment 4, pFFs were ultrafiltered, and separated into fractions greater and less than 30 kDa; the effects of the separated pFFs on nuclear maturation were examined. We used Student&amp;apos;s t-test to examine the differences among the results; a P value less than 0.05 was considered to be significant. In experiment 1, the progression of nuclear maturation was significantly faster when the maturation medium was supplemented with LpFF than with SpFF (MI rates at 36 h after culture: LpFF, 20.0%, and SpFF, 35.0%; MII rates at 36 h after culture: LpFF, 33.3%, and SpFF, 20.0%). The rates of polyspermic fertilization were significantly lower in the maturation medium with LpFF than in that with SpFF (LpFF, 31.7%; SpFF, 66.7%). Moreover, the rates of monospermic fertilization (LpFF, 45.0%; SpFF, 21.7%) and blastocyst formation (LpFF, 15.2%; SpFF, 3.0%) were significantly greater in the medium supplemented with LpFF than with SpFF. In experiment 2, we observed that LpFF accelerated the progression of nuclear maturation of pig oocytes collected from LFs (MII rates at 36 h after culture: LpFF, 61.7%; SpFF, 35.5%), but not those collected from SFs (MII rates at 36 h after culture: LFs, 15.0%; SFs, 11.7%). In experiment 3, the accelerating effect of pFF on nuclear maturation was eliminated by heat (MII rates at 36 h after culture: LpFF, 30.0%; SpFF, 25.0%) or trypsin treatment (MII rates at 36 h after culture: LpFF, 20.0%; SpFF, 21.7%). In experiment 4, only LpFF with a molecular weight greater than 30 kDa had positive effects on the progression of nuclear maturation (MII rates at 36 h after culture with LpFF: &amp;gt;30 kDa, 30.0%; &amp;lt;30 kDa, 11.7%). The present study suggests that LpFF contains some proteins that improve the progression of nuclear maturation and the developmental competence of oocytes.

Exposure to a Physiologically Relevant Elevated Temperature Hastens In Vitro Maturation in Bovine Oocytes

The objective of this study was to evaluate nuclear (progression to metaphase II) and cytoplasmic (translocation of cortical granules to the oolemma) maturation in control (38.5° C) and heat-stressed (41.0° C) oocytes. Hoechst staining indicated that a similar proportion of control and heat-stressed oocytes progressed to metaphase II. More heat-stressed oocytes had type III cortical granule distribution suggesting that heat stress accelerated cytoplasmic maturation. The kinetics of nuclear maturation was examined in a second experiment in which a higher proportion of heat-stressed oocytes progressed to metaphase I by 8h and arrested at metaphase II at 16 and 18h after placement into maturation medium. However, differences related to maturation temperature were no longer apparent by 21h. Heat-induced alterations in kinetics of nuclear and cytoplasmic maturation prompted a third experiment to evaluate if earlier insemination of heat-stressed oocytes ameliorates heat-induced reductions in development. A significant temperature × insemination time interaction was noted when evaluating blastocyst development. Blastocyst development was reduced when heat-stressed oocytes were inseminated with sperm 24h after placement into maturation medium compared with controls. In contrast, blastocyst development was similar to controls when heat-stressed oocytes were inseminated at 19h. Based on this interaction, earlier insemination in vitro prevented heat-induced reductions in oocyte development. Collectively, these studies suggest a cumulative effect of heat stress to hasten in vitro maturation in bovine oocytes.

Kinetics of nuclear maturation and effect of holding ovaries at room temperature on in vitro maturation of camel ( Camelus dromedarius) oocytes

Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4–48 h. At every 4 h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other ( P > 0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher ( P < 0.05) at 48 h compared with other maturation periods. There was no difference ( P > 0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12 h (43%, 64/148) and those collected in warm NSS (37 °C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups ( P < 0.05). It may be concluded that dromedary oocytes require 32–44 h of in vitro culture to have an optimum number of oocytes in M-II stage. However, further studies are required to find out the most appropriate maturation period, which will result in the further development of these oocytes after IVF, ICSI, parthenogenetic activation or nuclear transfer. Ovaries can be collected and stored in normal saline solution at room temperature for 12 h without any appreciable effect on the nuclear maturation of the oocytes.

274 NUCLEAR MATURATION KINETICS AND IN VITRO EMBRYO DEVELOPMENT OF BOVINE OOCYTES TREATED WITH BUTYROLACTONE I COMBINED OR NOT WITH ROSCOVITINE

Cyclin-dependent kinase inhibitors (CDKIs) have been used for prematuration culture the aim at improving oocyte competence. However, CDKIs seem to accelerate nuclear maturation (Hashimoto et al., 2002 Biol. Reprod. 66, 1696–1701). The aim of the present work was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at low dose (Ponderato et al, 2001 Mol. Reprod. Dev. 60, 579–585) on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 μM BLI (B) or 6.25 μM BLI + 12.5 μM ROS (BR) in TCM-199 for 24 h. After prematuration, oocytes were submitted to in vitro maturation (IVM in TCM-199 + 0.5 μg mL−1 FSH, 50 μg mL−1 LH, 10% FCS) for another 24 h. Oocytes were fixed every 3 h (40–50 oocytes/time point/group in 4 replicates) to assess nuclear status. In Experiment 2, oocytes were submitted to prematuration, but the inhibitors were diluted in TCM-199 or DMEM. IVM lasted 21 h in DMEM (same hormone supplementation as in TCM-199 + 5% FCS and 50 ng mL−1 EGF). After IVM, all groups (140–150 oocytes/group in 7 replicates) were in vitro fertilized. Oocytes and sperm (2 × 106 sperm cells mL−1) were co-cultured for 18 h. Embryos were cultured in CR2aa in co-culture with granulosa cells for 8 days. All cultures were in microdrops under oil, at 38.5°C under 5% CO2 in air. In both experiments, control oocytes (C) were submitted only to IVM. Data were analyzed by GLM and GENMOD procedures (SAS program; SAS Institute, Inc., Cary, NC, USA), for Experiments 1 (4 replicates) and 2 (7 replicates), respectively. Cell numbers were analyzed by ANOVA and Tukey test. In Experiment 1, at 0 h, C and B oocytes were all (100%) at germinal vesicle stage (GV). BR had less GV oocytes (89 ± 1%, P &lt; 0.05), indicating that BR was less effective in maintaining meiotic block for 24 h. After 3 h IVM, B and BR had less oocytes in GV (85 ± 2 and 80 ± 1%, respectively; P &gt; 0.05) than C (100%, P &lt; 0.05), suggesting an acceleration of oocyte maturation. At 12 h, however, most oocytes were at intermediate stages (metaphase I to telophase I) in all groups (78 ± 1–83 ± 2%, P &gt; 0.05). After 21 and 24 h, all groups had similar metaphase II (MII) rates (77 ± 1–89 ± 1 for 21 h and 85 ± 2–96 ± 8 for 24 h P &gt; 0.05). These results suggest that after 12 h, meiosis acceleration was less evident and oocytes proceeded nuclear maturation at similar rates. In Experiment 2, cleavage (79 ± 3–84 ± 3%, P &gt; 0.05) and Day 7 blastocyst rates (26 ± 4–37 ± 4%, P &gt; 0.05) were similar for all groups. After 8 days in culture, all groups presented similar blastocyst rates (35 ± 4–40 ± 4%, P &gt; 0.05), except for the group prematured with BR in DMEM, which presented lower blastocyst rates (32.3 ± 4%) only when compared with C (40 ± 4%, P &lt; 0.05). Hatching rates were similar (10 ± 3–16 ± %3, P &gt; 0.05) as were total cell numbers (141 ± 5–170 ± 10). In conclusion: (a) BR is less effective in maintaining meiosis block; (b) B and BR accelerate the first half of meiosis progression in about 3 h; and (c) BR used in DMEM during prematuration may negatively affect developmental rates. Financial support was provided by Fapesp, Brazil.

Effects of meiosis-inhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes.

Experiments were conducted to determine the effects of meiosis-inhibiting-agents and gonadotropins on nuclear maturation of canine oocytes. The culture medium was TCM199 + 10 ng/ml epidermal growth factor supplemented with 25 microM beta-mercaptoethanol, 0.25 mM pyruvate, and 1.0 mM L-glutamine (Basal TCM). Initially, oocytes were cultured in Basal TCM alone or in Basal TCM + dibutylryl cyclic adenosine monophosphate (0.5, 1, 5, or 10 mM dbcAMP) for 24 hr. Dibutylryl cAMP inhibited resumption of meiosis in a dose-dependent manner; 60% of oocytes remained at the germinal vesicle (GV) stage after being cultured for 24 hr in 5 mM dbcAMP. The meiosis-inhibitory effect of dbcAMP appeared to be reversible, as the oocytes resumed meiosis and completed nuclear maturation after being cultured for an additional 48 hr in its absence. Oocytes were then cultured in Basal TCM alone or in Basal TCM + roscovitine (12.5, 25, or 50 microM) for 24 hr. Although approximately 60% of oocytes cultured in 25 microM roscovitine remained at the GV stage, this percentage was not significantly different from the 48% that also remained at the GV stage when cultured in its absence. Oocytes were cultured in Basal TCM + 25 microM roscovitine for 17 hr, exposed briefly to equine chorionic gonadotropin (eCG), and then cultured in Basal TCM for 48 hr. Short exposure of oocytes to eCG was beneficial, as it significantly increased the proportion of oocytes developing beyond germinal vesicle breakdown (P < 0.05) with approximately 20-30% of these were metaphase I (MI) oocytes. Study of the kinetics of nuclear maturation demonstrated that large numbers of oocytes remained at MI even after being cultured for 52 hr following brief exposure to eCG. This study showed that in vitro maturation of canine oocytes can be somewhat improved by short exposure of oocytes to eCG. However, further studies are still required to derive effective methods to mature canine oocytes in vitro.

Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages

The kinetics of nuclear maturation, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes were examined. A further objective was to determine the duration of the meiotic stages during the maturation process. Porcine and bovine cumulus–oocyte complexes (COCs) were incubated in TCM 199 supplemented with 20% (v/v) heat inactivated fetal calf serum (FCS), 0.05 μg/ml gentamycin, 0.02 mg/ml insulin, 2.5 μg/ml FSH and 5 μg/ml LH. COCs were removed from the culture media in hourly intervals starting immediately after recovery from the follicle until 24 (bovine) or 48 h (porcine) of culture. Oocytes were either fixed to evaluate the maturation status or the activity of MPF, assessed by its histone H1 kinase activity, and MAP kinase were determined by a radioactive assay simultaneously. In oocytes of both species, the MPF activity oscillated during the culture period with two maxima corresponding with the two metaphases: between 27–32 and after 46 h (porcine) and between 6–9 and after 22 h (bovine). There was a temporary decline in activity after 33–38 (porcine) and after 19 h (bovine), which corresponded with anaphase I and telophase I. MAP kinase activity increased during the whole culture period and reached maximum levels after 47 (porcine) and after 22 h (bovine). In porcine oocytes, the MAP kinase was activated before GVBD and MPF activation. In bovine oocytes, MPF and MAP kinase were activated at approximately the same time as the GVBD (8–9 h of incubation). In average porcine, oocytes remain 23.4 h in the germinal vesicle (GV) stage (13 h in GV I, 5.7 h in GV II, 3.2 h in GV III and 1.5 h in GV IV), 0.9 h in diakinese, 9.6 h in the metaphase I, 2.8 h in anaphase I and 1.9 h in telophase I of the first meiotic division. In bovine oocytes, the temporal distribution of the meiotic stages were 8.5 h for the GV stage, 1.2 h for diakinese, 8.3 h for metaphase I, 1.6 h for anaphase I and 1.9 h for telophase I. These results indicate that the duration of the meiotic stages differs between the species and that MAP kinase is activated before MPF and GVBD in porcine oocytes.

The influence of cumulus-oocyte complex morphology and meiotic inhibitors on the kinetics of nuclear maturation in cattle

This study evaluated whether pre-established morphological classes of bovine cumulus oocyte complex (COCs) differ in their kinetics of meiosis resumption after 4 h of incubation and whether the timing of COCs resumption of meiosis differed after a period of maintained meiotic arrest. Bovine COCs were aspirated from 2 - to 5 - mm follicles and classified according to the state of their cumulus cells and cytoplasm (Classes 1 to 3). Groups of 15 to 20 COCs were fixed at 0 h or after an incubation period of 4 h. In addition, COCs from Class 1 were first incubated for 4 h on a theca cell monolayer or in the presence of 2 μg/mL of cycloheximide, rinsed and then incubated in cychloheximide and theca cell-free medium for another4 h. Oocytes then were fixed and evaluation for state of nuclear maturation. Results show that at 0 h, COCs from Class 3 have fewer oocytes at the GV stage than COCs from Class 1 and Class 2 (respectively 69.3 ± 3.2 vs 88.8 ± 3.4% and 86.9% GV ± 4.3% SEM; P < 0.05). After 4 h of incubation, all COCs classes show a significant decrease in the number of COCs at the GV stage. The COCs maintained in meiotic arrest and then incubated for 4 h resume meiosis faster than COCs incubated in cycloheximide and theca cell-free medium (19.4 ± 2.5, 33.3 ± 7.3 and 59.9 ± 6.5% GV SEM, respectively). The COCs of Class 3 have fewer oocytes at the GV stage at the beginning of incubation than all other classes. The number of COCs at the GV stage after 4 h of incubation in cycloheximide and theca cell-free medium is not significantly different than those COCs incubated in the presence of theca cell monolayers for 24 h (58.8 ± 6.5 vs. 56. 4 ± 6.4% SEM; respectively). Our results indicate that the ability of theca cells to maintain oocytes at the GV stage could be limited to those oocytes that were not committed or primed in vivo to resume maturation as indicated by their faster maturation kinetics.

Regulation of human and mouse oocyte maturation in vitro with 6-dimethylaminopurine.

It has been postulated that premature shortening of the oocyte growth phase due to the recovery of oocytes from small diameter follicles may be responsible for the developmental anomalies associated with in-vitro maturation. 6-Dimethylaminopurine (DMAP) was used to artificially lengthen the pre-maturation period of oocyte growth, in vitro, by inhibiting germinal vesicle breakdown in mouse and human oocytes. DMAP inhibited the meiotic maturation of mouse and human oocytes and the inhibition was fully reversible. The timing of polar body extrusion was accelerated in mouse oocytes following the withdrawal of DMAP; however, the kinetics of nuclear maturation in human oocytes was unaffected by exposure to DMAP. All mouse and human DMAP-treated oocytes that matured to metaphase II expressed histone H1 kinase activity. Fertilization rates in both DMAP-treated and control mouse and human oocytes were comparable, and human embryonic development was similar in control and DMAP-treated oocytes. However, blastocyst development was significantly reduced in DMAP-treated mouse oocytes (P < 0.05). It is concluded that lengthening the prematuration growth phase, by temporarily inhibiting kinase activity with DMAP, does not directly improve oocyte developmental competence but provides a useful tool for further investigating meiotic and developmentally related events in vitro by manipulating meiotic resumption.

Kinetics of nuclear maturation and protein profiles of oocytes from prepubertal and adult cattle during in vitro maturation

The aim of this present study was to compare the kinetics of nuclear maturation between calf and cow oocytes in order to determine if there are differences between the 2 groups which could explain their disparate developmental capacity. The constitutive and neosynthetic protein patterns of cow and calf oocytes and of their corresponding cumulus cells were also compared during in vitro maturation. A total of 397 calf oocytes and 406 cow oocytes was matured in M199 + 10 ng/mL EGF. The first group of oocytes (n=30) was immediately fixed and stained after removal from the follicle, and represent 0 h. The remaining oocytes were removed from the maturation medium at 4, 8, 12, 16, 20 and 24 h respectively. Half were denuded, fixed and stained for nuclear status; while the remainder were radiolabeled with methionine-( 35S). Immediately after isolation, all the oocytes were at the GV stage. By 8 h, GVBD had occurred in most oocytes (calf: 97%; cow: 100%) and some had reached pro-metaphase I (calf: 49%; cow: 51%). By 12 h, most of the oocytes were at metaphase I (calf: 84%; cow: 94%). By 16 h, 54% of calf oocytes had reached telophase I or beyond compared with 71% of cow oocytes. This difference between the 2 groups became significant by 20 h, with 89% of cow oocytes (P<0.05) at metaphase II and 71% of calf oocytes. By 24 h of culture, GVBD had occurred in all cases. Most oocytes completed meiosis I and were arrested at metaphase II with the first polar body extruded (calf: 72%; cow: 86%). No differences were noted in the constitutive and the neosynthetic protein profiles of cumulus cells in relation to the age of animal. Changes in neosynthetic protein patterns were observed both in cow and calf cumulus during IVM, and several proteins showed stage-specific synthesis. For the constitutive protein patterns of cow and calf oocytes, there were quantitative (38 and 40 kD) and qualitative (4, 10, 16, 17, 24, 25 and 26 kD) differences between the 2 groups. Only a few differences were observed in neosynthetic proteins between cow and calf oocytes, but there were changes in relation to nuclear status both in cow and calf oocytes. In conclusion, the difference in developmental capacity between cow and calf oocytes may be explained by a difference in the kinetics of nuclear maturation, which was significant at 20 h of culture (with 89% of cow oocytes at metaphase II and 71% of calf oocytes). At the biochemical level, our results indicate that nuclear progression during in vitro maturation of bovine oocytes is linked to changes in protein synthesis by the oocyte itself, while cumulus protein synthesis may either stimulate or modulate the process of oocyte maturation.