Effect of cycloheximide upon maturation of bovine oocytes

Germinal vesicle breakdown (GVBD) of bovine oocytes was completely blocked by cycloheximide added to culture medium at concentrations of 1-20 micrograms/ml. Nevertheless, under such conditions a certain degree of chromatin condensation inside the germinal vesicle was observed. The inhibitory effect was not influenced by the presence or absence of cumulus cells and was fully reversible; but the process of GVBD was then significantly accelerated. The critical period in which the proteins necessary for GVBD are synthesized lasts approximately the first 5 h of culture. When germinal vesicle-arrested oocytes are fused to maturing bovine oocytes containing condensed chromosomes, GVBD of immature oocytes occurs within 3 h, even in the presence of cycloheximide. In the mouse, GVBD cannot be inhibited by protein synthesis inhibitors. When immature mouse oocytes are fused with immature bovine oocytes and the giant cells are then cultured in cycloheximide-supplemented medium, both GVs are observed, or only mouse GVBD occurs in common cytoplasm after 8 h of culture. We conclude that protein synthesis is necessary for GVBD of bovine oocytes. Our results also suggest that maturation-promoting factor (MPF) is not autocatalytically amplified in mammalian oocytes.     

Cytoplasmic control of nuclear maturation in mouse oocytes

Oocytes with germinal vesicles were cut into anucleate and nucleate fragments. At the time of germinal vesicle breakdown (GVBD) in nucleate fragments (after 2–3 h of culture) sister anucleate fragments were fused with the help of inactivated Sendai virus with interphase blastomeres from 2-cell embryos. The hybrid cells were examined after h and 20 h. The anucleate fragments induced chromosome condensation in the nuclei of interphase blastomeres immediately after fusion. On this basis it may be concluded that GVBD and nuclear maturation in mouse oocytes is induced by a cytoplasmic factor which is produced or unmasked independently of the nucleus.     

Role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes

The role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes was investigated in the present study. To determine the estradiol effect, oocytes were cultured for 42 h in a steroid free medium composed of mTCM-199 supplemented with LH, FSH and 10% charcoal extracted follicular fluid. Estradiol receptor (ER), detected by a binding assay, were present in cumulus cells and oocytes during maturation with higher levels observed at 24 h of culture in the oocytes and at 36 h in the cumulus cells. To block estradiol action an antiestrogen (1-p-dimethylaminoethoxyphenyl-1,2-diphenyl-1-butene (tamoxifen)) was added to the maturation medium at various concentrations. The percentage of treated oocytes that underwent nuclear maturation was similar (P>0.05) to the control group. Cytoplasmic maturation, determined by the ability to form female pronucleus (FPN) and male pronucleus (MPN), was not different (P>0.05) among all groups. The presence of 4-hydroxy-4-androstene-3-17-dione (4-OHA) also did not influence nuclear (P>0.05) or cytoplasmic maturation (P>0.05). The results suggest that estradiol is not involved in maturation of pig oocytes. However, the present experiment used pronuclei formation as the endpoint, no studies were done in regard to estradiol's effects on the embryonic development.     

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 duration of in vitro maturation on nuclear maturation and fertilizability of feline oocytes

This study was conducted to improve in vitro production of embryos from domestic cats using TCM-199 as an IVM medium. The time sequence of nuclear maturation and the optimal timing of in vitro insemination were examined. Most oocytes were at the germinal vesicle stage immediately after collection; however, 8.3% had already resumed meiosis before IVM culture. After 30 h of IVM culture, the percentage of oocytes at metaphase II (MII) reached a peak (75.5%) and did not change (P>0.05) from 30 to 48 h after IVM culture. The percentage of oocytes with two pronuclei was higher (P<0.05) for oocytes matured for 30 and 36 h (38.2 and 33.0%, respectively) than for those after IVM culture for only 24 h (18.5%). Total sperm penetration rate was highest (P<0.05) for oocytes that had been matured for 30 h (46.1%). After 30 h of IVM and 18 h of IVF culture, 66.3 and 24.8% of inseminated oocytes had cleaved and developed to the blastocyst stage, respectively. We concluded that IVM of feline oocytes for 30 h in TCM-199 resulted in optimal nuclear maturation and sperm penetration.     

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2-3h after collection. Cumulus-oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4-6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4-6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.     

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

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

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

Effect of proteasome inhibitor MG132 on in vitro maturation of pig oocytes

The present study aimed to demonstrate the dependence of meiotic maturation in pig oocytes on the activity of the protease complex proteasome. The proteasome inhibitor MG132 blocked the exit of maturing pig oocytes from metaphase I stage. Seventy-five per cent of the oocytes were blocked at metaphase I when they were cultured with 10 microM MG132. The blocking effect of MG132 was expressed only when the oocytes were exposed to an inhibitor before the 18th hour of in vitro culture. The effects of MG132 are fully reversible. However, a significant proportion of oocytes (46%) cultured for 48 h in MG132-supplemented medium and then for 24 h in MG132-free medium did not block meiosis at the stage of metaphase II and underwent spontaneous parthenogenetic activation. On the basis of our data we can conclude that exit from the metaphase I stage of meiosis is proteasome-dependent in pig oocytes matured in vitro. On the other hand, our data also indicate that other proteasome-independent events are involved in regulating the exit from metaphase I.     

Inhibition of nuclear maturation in fully grown porcine and mouse oocytes after their fusion with growing porcine oocytes

Porcine ovarian oocytes, isolated from follicles of 5 mm in diameter (large oocytes), were fused either together or with oocytes isolated from follicles of 0.5 mm in diameter (small oocytes). In giant cells composed of two large oocytes (control) germinal vesicle breakdown (GVBD) occurred and two metaphase I chromosome sets (M I) were observed 24 to 30 h after fusion. By contrast, in giant cells composed of one large and one small porcine oocyte, both germinal vesicles (GVs) remained well conserved after 24-30 h of culture. An identical situation was observed after fusion and cultivation of small porcine and large mouse oocytes isolated from preovulatory follicles. The results demonstrate the presence of inhibiting activity in the ooplasm of small porcine oocytes that prevents nuclear maturation of large porcine and mouse oocytes fused to them. This maturation inhibiting activity can be overcome by preincubating large porcine oocytes for more than 14 h before fusion with small oocytes. During preincubation the ooplasm produces sufficient amount of maturation promoting factor (MPF) to overcome the inhibiting activity present in small porcine oocytes thus inducing GVBD and chromatin condensation both in small and large oocytes.     

Effect of follicle-stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro

A series of experiments were conducted to evaluate the effects of FSH supplementation during IVM on porcine oocyte nuclear maturation, and subsequent fertilization, cleavage and embryo development. Cumulus-oocyte complexes (COCs) were cultured 40 h without FSH (control), 40 h with FSH (FSH 0-40 h), or 20 h with FSH followed by a 20-h culture period without FSH (FSH 0-20 h). Nuclear stage of oocytes was assessed at intervals from 12 to 40 h of IVM. Furthermore, oocytes were in vitro fertilized, fixed and stained to determine normally fertilized and polyspermic oocytes. Additionally, COCs were matured with FSH, fertilized and zygotes cultured in NCSU-23. The percentage of cleaved embryos and blastocysts were determined and the number of nuclei was counted. The presence of FSH during the first 20 h of IVM retarded germinal vesicle breakdown. After 40 h of culture 84, 67 and 58% MII oocytes were observed in the FSH 0-20 h, FSH 0-40 h and control groups, respectively. After IVF, penetration rates were similar at 27, 26 and 29%, while the proportion of polyspermic oocytes was 7, 19 and 11% of penetrated oocytes for control, FSH 0-40 and FSH 0-20 h groups, respectively. Cleavage and blastocyst rates differed among treatments (21, 29 and 38%, and 7, 15 and 20% for control, FSH 0-40 and FSH 0-20 h groups, respectively). No differences in blastocyst cell number were found among groups. Blastocyst rates, based on number of cleaved embryos, were 51 and 52% for the FSH 0-40 and FSH 0-20 h groups, which differed significantly from the control group (31%). The results indicate that FSH has a stimulatory effect on nuclear and cytoplasmic maturation of sow oocytes. Addition of FSH for the first 20 h of culture was most beneficial, based on cleavage and blastocyst development rates.     

Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression.

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.     

Effect of phorbol ester PMA and puromycin on the maturation of mouse oocytes.

The effect of phorbol myristate acetate (PMA) and puromycin (PU) on maturation divisions of mouse oocytes was investigated. PMA changed the morphology and function of the spindle in meiosis I. The spindle had a broad polar region and did not commence karyokinesis in the first maturation division. This persisted in 25% of spindles which were passive up to 30 h of maturation. Half of the oocytes with spindles blocked by PMA between 20 and 30 h of maturation became activated, and 25% of the initial pool underwent abortive karyokinesis consisting of segregation of several chromosomes of the metaphase plate. Cytological observation indicated that the presence of PMA interfered with the elongation of the spindle in anaphase. Under the condition of changed phosphorylation of maturation of proteins by PMA, a reversible interphase was induced by PU. Most of the nuclei induced by PU exhibited pronuclei. A new class of nuclei induced by PU with condensed chromatin and having no nucleoli is described. The mechanism and consequences of interference of PMA on anaphase are discussed.     

Sperm penetration into immature mouse oocytes and nuclear changes during maturation: an EM study

The ultrastructure of oocyte and sperm nuclei was studied in mouse ovarian oocytes inseminated in vitro and cultured for 1 1/2 and 3 h in a medium containing dbcAMP or lacking the maturation inhibitor. In oocytes blocked at the germinal vesicle (GV) stage, certain maturation-linked changes were noted. Sperm apposition and sperm-oocyte fusion were similar to that during fertilization of ovulated oocytes. The sperm nucleus and its nuclear envelope remained intact after penetrating into the ovarian oocyte. One and a half h after removal of the drug (time 0 of maturation) the germinal vesicle (GV) and sperm nucleus remained intact. In oocytes maturing for 3 h, the nuclear envelopes of the GV and sperm nucleus had fragmented. The NE of the oocyte formed quadruple membranes while the NE of the sperm remained as flat vesicles. Oocyte chromatin condensed to form chromosomes, whereas at the same time the sperm chromatin was in the process of decondensation and was surrounded by fragments of the sperm NE. The sperm chromatin, composed of DNA complexed with protamines, consisted of thin fibrils; the individual fibrils measured 3.8 nm in diameter. Near the penetrated spermatozoa only occasional Mts were detected which were not related to the proximal centriole which was recognizable in the neck-piece of the flagellum. Thus in mouse oocytes the introduced sperm centriole is not capable of behaving as a centrosome and organizing microtubules in the form of an aster.     

Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro.

The present study was carried out to examine the effect of maturation media on male pronucleus formation of pig oocyte matured and fertilized in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughter house were cultured (36 h) in three different media (mTCM-199, Waymouth MB 752/l, and mTLP-PVA), fertilized in vitro, and assessed for nuclear maturation and male pronucleus formation. The addition of 10% (v/v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (P less than 0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 752/l with or without pFF than in oocytes matured in the other two media (P less than 0.01). In experiment 2, the addition of cysteine (the same concentration as in Waymouth medium, 0.57 mM), to mTLP-PVA significantly increased the rate of male pronucleus formation of pig oocytes compared with the control (P less than 0.01). The results indicate that the composition of maturation medium affects the ability of pig oocytes to form male pronuclei following sperm penetration; media containing a high concentration of cysteine (possibly as a substrate of glutathione), such as Waymouth MB 752/l, can remarkably promote this ability.     

Effects of different serum supplements in maturation medium on meiotic and cytoplasmic maturation of pig oocytes

The temporal progression of meiotic and cytoplasmic maturation of pig oocytes cultured in a medium supplemented with 0.4% polyvinylalcohol (PVA), 10% fetal calf serum (FCS), 10% newborn piglet serum (NPS), 10% porcine follicular fluid (PFF) or 10% porcine seminal fluid (PSF) was examined after 20, 30, 40 and 50 hours of culture. There were no differences in germinal vesicle breakdown (GVBD) among FCS and NPS supplements. After 20 hours of culture, the frequency for GVBD was higher (P < 0.05) in FCS and NPS (54% and 52%, respectively) than in PVA (32%) and PFF (33%) culture media but were not different at 40 and 50 hours of culture. Supplementation with PSF resulted in a rapid chromosome condensation of pig oocytes after 20 hours culture, but all GVBD oocytes stopped developing at the condensed germinal vesicle stage. Oocytes were not penetrated by spermatozoa when inseminated following 20 hours of culture, while high penetration (87 to 100%) and polyspermy rates (86 to 100%) were consistently obtained in all the supplement groups when inseminated after 30, 40 or 50 hours of culture. Male pronuclear formation rates at 10 to 12 hours after insemination, following a 50-hour culture period in FCS and NPS, were 28 and 28%, respectively, in comparison with 54% in PVA and 59% in PFF. The results indicate that supplementing maturation media with serum such as FCS and NPS reduced the ability of pig oocytes to form a male pronucleus, and further suggest that the detrimental effects may be due to accelerated progression of maturation events.     

Heparin effect on in vitro nuclear maturation of bovine oocytes

Oocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized. Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 degrees C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7+/-1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0+/-1.1 h to 9.0-11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.     

Involvement of steroid hormones on in vitro maturation of pig oocytes

The purpose of this study was to determine if the addition of steroid hormones into the culture medium could influence the in vitro maturation of pig oocytes. The cumulus-oocyte complexes (COCs). collected from follicles of 2-5 mm diameter, were matured in steroid-free medium supplemented with various concentrations of estradiol-17beta (0-3000 ng/ml), progesterone (0-5000 ng/ml) and testosterone (0-300 ng/ml). The COCs were cultured for 42 h, then fertilized in vitro. We analyzed nuclear and cytoplasmic maturation with lacmoid stain 20 h after in vitro insemination. We observed no significant effect (P > 0.05) on the percentage of oocytes completing nuclear or cytoplasmic maturation or the number of sperm penetrating each oocyte for any concentration of progesterone, estradiol-17beta or testosterone. Similarly, adding a combination of those hormones to the medium did not significantly (P > 0.05) affect any of the criteria. In order to determine if there was a possible secretion of steroids during maturation, we added COCs, denuded oocytes and stripped cumulus cells to drops of a steroid-free medium and cultured them for 42 h, after which we analyzed the medium, before and after culture, for the presence of progesterone, estradiol-17beta and testosterone by radioimmunoassay (RIA) analysis. COCs, as well as cumulus cells alone, secreted similar amounts of estradiol (43.3 and 37.5 pg/ml, respectively) and progesterone (4.24 and 4.79 ng/ml, respectively) into the maturation medium. A small amount of estradiol (28.8 pg/ml) was also detected when oocytes were cultured alone. These results indicate that no steroids need to be added to the maturation medium of pig oocytes and that the COCs secrete steroids during maturation. It is possible that the amounts produced by the COCs fulfill any requirement for steroids if these steroids are required for either nuclear or cytoplasmic oocyte maturation.     

Protein patterns of pig oocytes during in vitro maturation

In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.     

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.